GIFT  OF 


t 


UNIVERSITY  OF  CALIFORNIA. 


FINAL  EXAMINATION 


OF 


VICTOR    BIRCKNER 


M.S.,   (UNIVERSITY  OF  CALIFORNIA)   1911 


FOR  THE 


DEGREE  OF  DOCTOR  OF  PHILOSOPHY, 


MONDAY,   APRIL    29,    1912, 


AT  2:00  P.M.,  IN  THE  RUDOLPH  SPRECKELS  PHYSIOLOGICAL  LABORATORY. 


SUB-COMMITTEE  IN   CHARGE: 

PROFESSOR  T.   BRAILSFORD  ROBERTSON. 
PROFESSOR  W.   A.   SETCHELL. 

PROFESSOR  A.  R.  MOORE. 

PROFESSOR  S.   S.   MAXWELL. 

PROFESSOR  w.  L.  JEPSON. 


(  Physiological  Chemistry. 
MAJOR  SUBJECT  < 

(  Research  Work  on  Sugar  Fermentations. 


I.  Advanced  Chemical  Biology.     Professor  Robertson. 
II.  Research  Work  in  Physiological  Chemistry.    Professor  Robertson. 

MINOR  SUBJECT:  BOTANY 

I.  A  general  study  of  the  orders  of  the  spore-bearing  green  plants  from  the  points  of  view  of 
structure,  development,  and  economic  importance.     Professor  Setchell. 

II.  Experimental  Plant  Physiology.     Research  work  on  some  factors  influencing  the  germina- 
tion of  seeds.    Professor  Moore. 

OTHER  SUBJECTS 

I.  Research   in   Chemico-Agricultural   Technology.    Professor  Shaw. 
II.  Lectures  on  Dairy  Chemistry.     Professor  Jaffa  and  Mr.  McCharles. 

III.  Advanced  work  in  the  Chemical  and  Microscopical  investigation  of  Fertilizers.      Professor 

Burd. 

IV.  Water  Supply  for  Irrigation.     Conservation  and  Use  of  Water.     Professor  Chandler. 


THESIS  : — On  the  Oxydations  and  Cleavages  of  Glucose. — Yeast  Glucase,  a  new 
Glucolytic  Ferment. 


SUMMARY 

(1)  A  systematic  review  has  been  given  in  the  first  part  of  the  paper  of  some  recent  advances 
of  our  knowledge  of  glucose  oxydations  and  cleavages,  both  outside  and  inside  of  the  organism. 

(2)  In  the  second  part  of  this  article  a  ferment  has  been  described  which  occurs  in  the 
California  steam  beer  yeast  under  certain  conditions,  and  which  has  the  property  of  accelerating 
the  decomposition  of  glucose  at  an  elevated  temperature. 

(3)  This  new  ferment  is  not  identical  with  zymase.     It  acts  preferably  at.  a  temperature  <>!' 
70°  C.    It  causes  no  gas  formation,  and  yields  no  alcohol. 

(4)  Its  action  on  glucose  manifests  itself  by  a  rapid  darkening  of  the  mixture,  a  strongly 
acid  reaction,  a  gradual  formation  of  a  carbon-like,  solid  settlement,  and  the  development  of  an 
odor  similar  to  caramel. 

(5)  The  ferment  may  be  extracted  from  a  yeast  powder  (Dauerhewe)  which  is  best  obtained 
by  treating  the  cells  with  aethyl-alcohol. 

(6)  From  the  watery  extract  the  yeast  glucase  may  be  obtained  and  purified  by  repeated 
precipitation  with  alcohol;  but  this  process  always  involves  a  weakening  of  the  ferment. 

(7)  Yeast  glucase  is -very  stable  in  aqueous  solution  if  kept  at  room  temperature  under 
sterile  conditions.    Boiling  for  one  minute  does  not  destroy  its  activity. 

(8)  Yeast  glucase  shows  activity  in  neutral  or  acid  solution   against  glucose,   polyphenols, 
and  lactates.     The  preparation  does  not  contain  tyrosinase,  nor  does   it   act   as  a   peroxydase 
against  glucose. 

(9)  The  ferment  preparation  gives  a  strong  pyrrol  reaction  (Neuberg). 

(10)  Yeast  glucase  shows  some  relationship  to  the  oxydases,  but  with  regard  to  its  main 
function,  it  is  to  be  classed  together  with  zymase  in  a  group  which  stands  separately  from  the 
oxydases  and  the  hydrolytic  ferments,  and  to  which  £uler  has  applied  the  names  "G-arungs 
fermente." 

(11)  The  transformation  products  of  glucose,  resulting  from  the  action  of  this  ferment,  are 
largely  acids,  none  of  which  has  so  far  been  clearly  identified.     However,  among  the  cleavage 
products  of  the  sugar,  the  presence  of  pentose  and  formaldehyde  was  ascertained,  which  is  of 
interest  with  regard  to  the  recent  work  of  W.  Lob. 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS 

'•  Vl|*,; 
PHYSIOLOGY 

Vol.  4,  No.  16,  pp.  115-183  September  20,  1912 


ON  THE  OXYDATIONS  AND  CLEAVAGES 

OF  GLUCOSE.     YEAST  GLUCASE, 

A  NEW  GLUCOLYTIC  FERMENT 


BY 

VICTOR  BIRCKNER 


UNIVERSITY  OF  CALIFORNIA  PRESS 
BERKELEY 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS 

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OTTO  HAREASSOWITZ,  R.  FRIEDLAENDER  &  SOHN, 

LEIPZIG.  BERLIN. 

Agent  for  the  series  in  American  Arcn-  Agent  for  the  series  in  American  Arch- 
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PHYSIOLOGY — S.  S.  Maxwell,  Editor.    Price  per  volume  $2. 

Cited  as  Univ.  Calif.  Publ.  Physiol. 

VoLl.      1.  On  a  Method  by  which  the  Eggs  of  a  Sea-urchin  (Str»ngylocentrotu» 
purpuratus)  can  be  Fertilized  with  the  Sperm  of  a  Starfish  (Atteriat 

ochracea),  by  Jacques  Loeb.    Pp.  1-3.    April,  1903._ 05 

2.  On  the  Mechanism  of  the  Action  of  Saline  Purgatives,   and  the 
Counteraction  of  their  Effect  by  Calcium,  by  John  Bruce  Mac- 

Callum.     Pp.  6-6.     May,  1903 05 

S.  Artificial  Parthenogenesis  in  Molluscs,  by  Jacques  Loeb.     Pp.  7-9. 

August,  1903  - _      .05 

4.  The  Relations  of  Biology  and  the  Neighboring  Sciences,  by  Wilhelm 

Ostwald.    Pp.  11-31.    October,  1903 25 

5.  The  Limitations  of  Biological  Research,  by  Jacques  Loeb.    Pp.  33-37. 

October,   1903   __ 05 

6.  The  Fertilization  of  the  Egg  of  the  Sea-urchin  by  the  Sperm  of  the 

Starfish,  by  Jacques  Loeb.    Pp.  39-53.    November,  1903 15 

7.  On  the  Relative  Toxicity  of  Distilled  Water,  Sugar  Solutions  and 

Solutions  of  the  various  Constituents  of  the  Sea-water  for  Marine 
Animals,  by  Jacques  Loeb.    Pp.  55-69.    November,  1903. 

8.  On  the  Segmental  Character  of  the  Respiratory  Center  in  the  Medulla 

Oblongata  of  Mammals,  by  Jacques  Loeb.    Pp.  71-75.    November, 
1903. 
Nos.  7  and  8  in  one  cover .25 

9.  On  the  Production  and  Suppression  of  Glycosuria  in  Rabbits  through 

Electrolytes  (a  preliminary  communication),  by  Martin  H.  Fischer. 

Pp.  77-79.     December,   190SL- .'. ~ _..      .05 

10.  On  the  Influence  of  Calcium  and  Barium  on  the  Flow  of  Urine  (a 

preliminary  communication),  by  John  Bruce  MacCallum.    Pp.  81-82. 
January,  1904 .05 

11.  Further  Experiments  on  the  Fertilization  of  the  Egg  of  the  Sea-urchin 

with  Sperm  of  various  species  of  Starfish  and  a  Hclothurian,  by 
Jacques  Loeb.     Pp.  83-85.     February,  1904 .05 

12.  On  the  Production  aud  Suppression  of  Glycosuria  in  Rabbits  through 

Electro.\Ftos    (sepoijd,  , communication),    by    Martin    H.    Fischer. 

Pp.  87-'i;i£..V  Febr>o&rjr,:  rt;3G4.; 30 

13.  The  Influence  of  Saline  "Purgatives  on  Loops  of  Intestine  Removed 

from,  tfcp  'B$ifcr?  bjr'J-ohik  Bruce  MacCallum.    Pp.  115-123.    March, 
1903-.'.  :;•'','•;          ;  ;    ;•  .  '.  , 

14.  The  Secretion  of  Sugar  into  the  Intestine  Caused  by  Intravenous 

Saline  Infusions,  by  John  Bruce  MacCallum.    Pp.  125-137.    March, 
1904. 
Nos.  IS  and  14  in  one  cover 20 

15.  On  the  Influence  of  the  Reaction  of  the  Sea-water  on  the  Regeneration 

and  Growth  of  Tubularians,  by  Jacques  Loeb.    Pp.  139-147.    April, 

1904   _ .10 

16.  The  Possible  Influence  of  the  Amphoteric  Reaction  of  Certain  Colloids 

upon  the  Sign  of  their  Electrical  Charge  in  the  Presence  of  Acid 
and  Alkalis,  by  Jacques  Loeb.    Pp.  149-150.    May,  1904. 

17.  Concerning  Dynamic  Conditions  which  contribute  toward  the  Deter- 

mination of  the  Morphological  Polarity  of  Organisms   (first  com- 
munication),   by    Jacques    Loeb.      Pp.    151-161.     7    text    figures. 
May,  1904. 
Nos.  16  and  17  in  one  cover _ 16 

18.  The  Action  of  Cascara  Sagrada  (a  preliminary  communication),  by 

John  Bruce  MacCallum.    Pp.  163-164.    May,  1904 05 

19.  Artificial  Parthenogenesis  and  Regular  Segmentation  in  an  Annelid 

(Ophelia),  by  G.  Bullot.    13  text  figures.    Pp.  165-174.    June,  1S04-.      .10 

20.  On  the  Action  of  Saline  Purgatives  in  Rabbits  and  the  Counteraction 

of  their  Effect  by  Calcium  (second  communication),  by  John  Bruce 
MacCaUum.    Pp.  175-185.    July,  1904, 


UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

PHYSIOLOGY 

Vol.  4,  No.  16,  pp.  115-183  September  20,  1912 


ON  THE  OXYDATIONS  AND  CLEAVAGES  OF 

GLUCOSE.  YEAST  GLUCASE,  A  NEW 

GLUCOLYTIC  FERMENT* 

BY 

VICTOR   BIECKNEB 


CONTENTS 

PAGE 

Introduction H6 

Part  I.    Modern  Viewpoints  on  the  Mechanism  of  the  Oxydations  and 
cleavages   of   Glucose   Inside   and   Outside   of   the   Living 

Organism    119 

A.  Destruction  of  glucose  by  chemical  or  physical  agencies  of  known 

character    119 

1.  The  structure  of  the  glucose  molecule  119 

2.  The  amphoteric  character  of  the  sugars  122 

3.  The  action  of  radiant  energy  on  glucose  126 

(a)   The  electrolysis  of  sugars — Lob's  theory  126 

(5)   The  action  of  light  136 

4.  The  action  of  alkalies  and  acids  on  glucose  138 

B.  The  oxydations  and  cleavages  of  glucose  through  the  action  of 

more  or  less  unknown  agencies  144 

Part  II.     Yeast  Glucase,  a  New  Glucolytic  Ferment  158 

1.  The  ferment  as  first  observed  and  recognized  158 

2.  Experiments  with  hydroquinone  165 

3.  Method  of  preparing  the  yeast  powder  171 

4.  Method  of  extracting  the  ferment  172 

5.  Attempts  of  further  purification  172 

6.  Properties  of  the  yeast  extract  174 

7.  Studies  on  the  products  of  glucose  fermentation  176 

Conclusions    .                        182 


*  Presented  in  partial  fulfillment  of  the  requirements  for  the  degree  of 
Doctor  of  Philosophy  at  the  University  of  California. 


244546 


•  .•  •»•  »•'*••*  •     »    «    •"*,, 
•*•••»»•*•      •   •*        1 

:•  s/:V  :!.,:>•,-.. 

116        University  of  California  Publications  in  Physiology  [VOL.  4 


INTRODUCTION 

The  work  of  Emil  Fischer1  on  the  artificial  synthesis  of 
sugars  in  1890,  marks  the  beginning  of  a  new  period  of  bio- 
chemical research.  The  foundations  of  an  accurate  study  of 
the  carbohydrates  were  laid. 

This  study  was  taken  up  immediately  by  a  large  number  of 
workers,  and  numerous  new  facts  were  brought  to  light  during 
the  last  twenty  years,  partly  through  Fischer's  own  efforts.  It 
is  therefore  not  surprising  that  our  conceptions  to-day  are 
already  slightly  beyond  the  original  conceptions  of  the  great 
investigator. 

The  carbohydrates  are  considered  as  being  among  the  most 
important  food  materials  of  the  animal  body.  They  form  a 
rather  uniform  class  of  substances  which,  especially  in  its 
lower  members,  is  well  understood  as  far  as  chemical  composi- 
tion and  properties  are  concerned.  We  are  confronted  by  the 
greatest  difficulties,  however,  when  trying  to  investigate  in  what 
way  these  substances  are  naturally  built  up  and  broken  down  in 
the  living  organism. 

The  central  figure  in  the  whole  carbohydrate  metabolism  of 
any  living  being  is  the  rather  simple  looking  substance  glucose, 
of  the  empirical  formula  C6H12OG.  This  substance  is  formed  in 
enormous  quantities  in  the  green  parts  of  the  plant  by  a  rapid 
reduction  of  the  carbon  dioxide  of  the  air,  and  subsequent 
polymerization  processes.  Through  further  condensations  of 
glucose  molecules,  the  plant  synthesizes  and  stores  up  at  the 
proper  places  those  immense  masses  of  reserve  materials,  to 
which,  either  directly  or  indirectly,  the  whole  animal  world  owes 
its  subsistence. 

On  its  way  through  the  animal  body,  the  complex  carbo- 
hydrate molecule  undergoes  changes  similar  to  those  by  which 
its  synthesis  was  brought  about  in  the  plant,  only  in  the  opposite 
direction.  The  molecule  is  first  broken  down  through  the  action 
of  certain  ferments  of  the  alimentary  canal  to  the  state  of 


1  Emil  Fischer,  several  articles  in  Ber.  DeutscJi.  chem.  Ges.,  vol.  23  and 
following;  collected  in  Untersuchungen  iiber  Kohlcnhydrate  und  Fermcnte 
(Berlin,  J.  Springer,  1884-1908). 


1912]  Bircknf.r:  Glucose  Oxydations  117 

glucose,  whereupon  resorption  by  the  blood  stream  can  take 
place.  After  thus  being  taken  into  the  circulating  system,  the 
glucose  molecule  undergoes  various  changes,  the  nature  of 
which  is  very  little  understood.  Part  of  the  sugar  we  find  again 
in  a  polmerized  form  as  glycogen  in  the  liver  and  in  the  muscles. 
Its  main  portion,  however,  serves  as  substrate  for  that  slow 
oxydation  (combustion)  process  which  is  constantly  going  on  in 
every  cell  of  the  body,  more  extensively  in  the  lungs,  and  the 
final  products  of  which  we  know  to  be  carbon  dioxyde  and  water. 

The  synthetic  part  of  this  carbon  cycle  (CO2  — >  glucose  -- •» 
polysaccharide)  is  almost  exclusively  a  function  of  the  green 
plant,  while  the  reverse  reaction  (polysaccharide  — »  glucose  — » 
COo)  is  constantly  going  on  in  both  plants  and  animals. 

The  ease  with  which  all  these  changes  are  apparently  brought 
about  in  the  living  cells  and  tissues,  at  a  rather  low  temperature 
and  in  a  practically  neutral  medium,  together  with  the  fact  that 
we-  have  a  perfectly  clear  understanding  of  the  physical  and 
chemical  properties  of  the  chief  substance  involved,  and  that  we 
can  prepare  this  substance  free  from  impurities,  have  been  a 
constant  stimulus  for  the  investigator  for  a  large  number  of 
years. 

Numerous  attempts  have  been  made  to  gain  an  insight  into 
the  ways  and  means  by  which  nature  brings  about  these  funda- 
mental transformations,  a  knowledge  of  which  would  mean  an 
important  step  towards  the  final  solution  of  the  problem  of 
artificial  synthesis  of  organic  matter  in  more  economic  ways. 

It  is  of  equal  importance  for  this  purpose  whether  we  study 
the  synthetic  process  as  such,  or  whether  we  try  to  follow  the 
glucose  molecule  as  it  is  being  broken  down  in  the  organism. 
The  latter  way  of  proceeding  is  perhaps  of  more  immediate 
interest  in  view  of  its  bearing  on  the  physiology  and  pathology 
of  our  own  body.  Progress  is  being  made,  however,  in  both 
directions,  and,  although  at  present  our  knowledge  on  either  side 
does  not  go  far  beyond  the  stage  of  a  hypothesis,  it  is  to  be 
noticed  after  the  advances  of  recent  years  that  the  workers  at 
both  sides  of  the  problem  are  approaching  each  other  more  and 
more  closely,  and  that  the  unexplored  area  between  them  is  being 
constantly  diminished. 


118        University  of  California  Publications  in  Physiology  [VOL.  4 

I  shall  try  in  the  first  part  of  this  paper  to  review  briefly  the 
more  recent  attempts  that  have  been  made  with  the  object  of 
approaching  the  problem  of  "  glucolysis. "  By  this  term,  which 
is  frequently  to  be  met  with  in  the  newer  literature,  may  be 
understood2  all  exothermic  cleavages  of  the  glucose  molecule 
which  furnish  energy  to  the  body,  no  matter  if  through  the 
interaction  of  oxygen,  or  in  its  absence.  In  some  English  articles3 
I  notice  the  word  being  spelled  "glycolysis"  in  analogy  to  the 
German  form.  As  this  term,  however,  is  apparently  already 
in  use  for  a  different  process,4  I  prefer  to  write  the  word 
' '  glucolysis. ' ' 


2  According  to  W.  Lob,  Beitrage  zur  Frage  der  Glycolyse   I,  Biochem. 
Zeitschrift,  vol.  29,  p.  317,  1910. 

3  E.g.,  L.  Henderson,  The  instability  of  glucose  at  the  temperature  and 
alkalinity  of  the  body,  Journ.  Biol.  Chem.,  vol.  10,  p.  4,  1911. 

4  Gould,  Medical  Dictionary,  p.  524,  194. 


1912]  Birckner:  Glucose  Oxydations  119 


PART  I 

MODERN  VIEWPOINTS  ON  THE  MECHANISM  OF  THE 
OXYDATIONS  AND  CLEAVAGES  OF  GLUCOSE 
INSIDE  AND  OUTSIDE  OF  THE  LIVING 
ORGANISM 

The  matter  which  is  being  taken  up  on  the  following  pages 
represents  the  first  trial,  to  my  knowledge,  to  bring  together  and 
arrange  systematically  all  important  experimental  results  of 
recent  years  which  have  helped  to  elucidate  somewhat  the 
phenomena  involved  in  the  decomposition  of  grape  sugar.  The 
subject  which  I  shall  try  to  cover  has,  undoubtedly,  not  yet 
arrived  at  the  stage  where  the  available  data  have  the  full  value 
of  facts,  and  it  would  hardly  pay  at  present  to  enter  into  a 
lengthy  discussion  of  all  experimental  results  obtained  and  of 
every  hypothesis  advanced.  Still,  as  stated,  the  achievements  of 
recent  years  are  rather  encouraging,  and  as  the  problem  is  of 
equal  importance  for  several  branches  of  science,  the  literature 
on  the  subject'  is  becoming  extensive,  the  problem  being  fre- 
quently treated  from  very  different  points  of  view.  For  a  person 
interested  anew  in  this  study  it  is  already  difficult  to  look  over 
the  entire  field;  and  being  aware  of  this  circumstance,  I  dare 
to  hope  that  the  following  sketch  of  the  present  stand  of  the 
question  will  not  be  useless. 

A.  DESTRUCTION  OF  GLUCOSE  BY  CHEMICAL  OR  PHYSICAL 
AGENCIES  OF  KNOWN  CHARACTER 

1.  The  Structure  of  the  Glucose  Molecule 

A  few  words  may  be  said  at  the  outset  with  regard  to  the 
configuration  formula  of  glucose,  as  a  more  intimate  inquiry 
into  the  phenomena  of  multirotation,  together  with  a  better 
understanding  of  the  properties  and  the  chemical  constitution  of 


120        University  of  California  Publications  in  Physiology  [VOL.  4 

the  glucosides,  have  brought  to  light  during  recent  years  many 
important  facts  which  for  various  reasons  demand  a  thorough 
revision  of  our  present  views,  and  to  which  the  biologist  should 
not  fail  to  pay  due  attention. 

As  the  first  oxydation  product  of  normal  hexane,  glucose  is 
usually  represented  by  the  formula  CH2(OH).  CH(OH). 
CH(OH).  CH(OH)  CH(OH).  CHO,  this  open-chain  formula 
bearing  an  aldehyde  group  at  one  end,  and  an  alkohol  radicle  at 
the  other. 

It  has,  however,  long  been  noticed  that  the  substance  is  far 
less  active  chemically  than  would  be  expected  from  a  hydroxy- 
aldehyde.  Tollens,1  therefore,  as  early  as  1883,  suggested  to 
express  this  relatively  high  stability  by  representing  the  mole- 
cule through  a  ring  formula,  the  ring  containing  four  of  the 
six  carbon  atoms  and  one  atom  of  oxygen.  As  a  result  of  the 
researches  of  Emil  Fischer,2  Tanret,3  E.  F.  Armstrong,4  and 
others5  on  the  two  stereoisomeric  forms  of  glucose  and  their 
relations  to  the  corresponding  methyl-glucosides,  the  glucose 
formula  which  is  now  almost  generally  agreed  upoii5a  is  the 
following : 


1 B.   Tollens,   Das   Verhalten   der   Dextrose   zu   ammoniakalischer   Silber- 
lo'sung,  Ber.  Deutsch.  chem.  Ges.,  vol.  16,  p.  921,  1883. 

2  E.  Fischer,  Einige  Sauren  der  Zuckergruppe,  Ber.  Deutscli.  chem.  Ges., 
vol.  23,  p.  2625,  1890;  tiber  die  Glucoside  der  Alkohole,  ibid.,  vol.  26,  p. 
2400,  1893;  ibid.,  vol.  28,  p.  1145,  1895. 

3  C.  Tanret,  Les  modifications  moleculaires  du  glucose,  Bull.  Soc.  Chim., 
(m),  vol.  13,  pp.  625,  728,  1895;  Compt.  rend.,  vol.  120,  pp.  1060-1062,  1895; 
Les  modifications  moleculaires  et  la  multirotation  des  sucres,  Bull.  Soc.  Chim., 
(m),  vol.  15,  pp.  195,  349,  1896;  Les  transformations  des  sucres  a  multirota- 
tion, Bull.  Soc.  Chim.  (m),  vol.  33,  p.  337,  1905. 

4  E.  F.  Armstrong,  Studies  on  Enzyme  Action.     I.  The  correlation  of  the 
stereoisomeric  a-  and  /3-glucosides  wtih  the  corresponding  glucoses,  Journ. 
Chem.  Soc.,  vol.  83,  p.  1305,  1903. 

5  E.g.,  L.  J.  Simon,  Sur  la  constitution  du  glucose,  Compt.  rend.,  vol.  132, 
pp.  487,  596,  1901.     For  details  and  other  literature  on  this  paragraph  see 
E.  F.  Armstrong,  The  Simple  Carbohydrates  and  the  Glucosides.  Monographs 
on  Biochemistry  (London,  Longmans,  Green  &  Co.,  1910),  p.  3  and  follow- 
ing, and  pp.  93-94. 

5a  See  for  instance  Emil  Fischer,  Ber.  d.  Deutsch.  chem.   Ges.,  vol.   45, 
p.  461,  1912. 


1912] 


Birckner:  Glucose  Oxydations 


121 


OH 


CH.  CH(OH).  CH2(OH) 


It  is  seen  that  in  this  closed-chain  formula  the  molecule  has 
five  asymmetric  carbon  atoms  as  against  four  in  the  empirical 
formula.  It  is  further  seen  that  the  carbon  atom  at  the  left  end 
of  the  pentaphane  ring  does  not  carry  an  aldehyde  group  any 
more.  As  a  matter  of  fact,  however,  glucose  in  aqueous  solution 
displays  aldehydic  properties.  To  account  for  this  behavior  it 
is  to  be  assumed  that  in  aqueous  solution,  part  of  the  molecules 
undergo  a  sort  of  intramolecular  hydrolysis,  by  which  the 
pentaphane  ring  is  ruptured  and  the  aldehyde  potential  of  the 
carbon  atom  to  the  left  set  free.  In  an  aqueous  solution  of 
glucose,  therefore,  according  to  E.  F.  Armstrong,6  the  following 
equilibrium  is  established : 


H       OH 

V 


HC(OH) 


-f  H,O 


O 


(OH)CH 


HC 
HC(OH) 

H2C(OH) 

Closed  Ring 

(»y-oxide 
modification) 


H       OH 

Y 


HC(OH)     OH 
(OH)CH  OH 

HC/ 

I 
HC(OH) 

H2C(OH) 

Aldehydrol 


O      H 

V 

HC(OH) 


OH 


HC(OH) 

H2C(OH) 

Aldehyde 
(open  chain 
modification) 


Supposing  now  the  aldehyde  be  attacked  by  an  oxydizing 
.agent  such  as  Fehling's  solution;  its  destruction  will  disturb 
the  equilibrium,  causing  the  heptahydric  alcohol  (aldehydrol), 
which  is  formed  intermediately,  to  change  into  the  aldehyde, 
and  this  again  causing  a  fresh  formation  of  aldehydrol  from  the 
closed-ring  form.  In  this  way,  as  the  reaction  proceeds,  all  of 
the  closed-ring  modification  will  be  finally  changed  into  aldehyde. 


E.  F.  Armstrong,  The  Simple  Carbohydrates,  etc.,  p.  4. 


122        University  of  California  Publications  in  Physiology  [VOL.  4 

On  the  other  hand,  if  a  substitution  or  esterification  product 
(such  as  a  glucoside)  is  to  be  formed,  the  reaction  will  take  place 
at  the  closed-ring  side  of  the  system,  and  all  the  aldehyde  will 
be  transformed  to  the  closed-ring  modification.  The  reaction, 
therefore,  is  a  reversible  one. 

Somewhat  similar  relations  are  assumed  by  Lowry7  to  under- 
lie the  isomeric  change  of  a-  into  /3-glucose  and  its  reverse.  To 
the  fact  that  in  aqueous  solution  glucose  always  exists  in  these 
two  stereo-isomeric  modifications,  which  form  an  equilibrated 
mixture  and  can  readily  be  converted  into  one  another,  E.  F. 
Armstrong8  attributes  considerable  biological  significance.  No 
experimental  data  are  at  present  available  with  regard  to  this 
point. 

2.  The  Amphoteric  Character  of  the  Sugars 

An  aqueous  solution  of  pure  glucose,  if  kept  under  sterile 
conditions,  shows  very  high  stability.  At  room  temperature 
it  may  be  kept  for  long  periods  before  the  solution  turns 
slightly  yellow,  or  before  a  fall  of  the  optical  activity  or  of  the 
copper-reducing  power  becomes  distinctly  noticeable.  At  "  a 
higher  temperature  these  changes  occur  more  rapidly,  as  the 
dissociation  of  the  water  itself  is  considerably  increased.  Still 
at  a  temperature  of  70°  C.  a  10  per  cent  solution  of  pure  glucose 
does  not  show  the  slightest  change  in  appearance  and  color  for 
at  least  three  weeks  (it  was  the  question  of  coloration  which  at 
the  beginning  of  my  work  interested  me  more  than  anything 
else,  as  will  be  seen  in  the  second  part  of  this  paper.) 

Experiments  of  Kullgren9  had  already  demonstrated  the 
strongly  depressing  effect  that  cane-sugar  had  on  the  velocity  of 
saponification  of  aethyl  acetate  by  sodium  hydroxide,  and  thereby 
established  the  acid  character  of  this  sugar  in  the  presence  of 
alkali. 

E.    Cohen10   on   repeating  these   experiments   with   different 


?  T.  M.  Lowry,  Studies  of  Dynamic  Isomersism.  The  mutarotation  of 
glucose,  Journ.  Chem.  Soc.,  vol.  75,  p.  213,  1899;  ibid.,  vol.  83,  p.  1314,  1903. 

s  E.  F.  Armstrong,  The  Simple  Carbohydrates,  etc.,  p.  20. 

»  Svensk.  Akad.  Handl.,  vol.  24,  1898 ;  cf .  E.  Cohen,  Studien  iiber  die 
Inversion,  Zeitschr.  f.  Physilc.  Chemie,  vol.  37,  p.  69,  1901. 

10  E.  Cohen,  loc.  cit. 


1912]  Birckner:  Glucose  Oxydations  123 

sugars  found  that  they  all  show  more  or  less  the  same  behavior. 
From  his  tables  (his  results  had  already  been  published  in 
Dutch  a  year  or  two  before)  Osaka11  succeeded  in  calculating 
the  dissociation-constant  of  glucose.  He  obtained  the  value  of 
5.9  X  10-13  at  25°  C. 

On  the  other  hand,  it  could  be  followed  from  the  work  of 
Baeyer  and  Villiger,12  Cohen,13  Walden,14  Walker,15  and  recently 
of  Stieglitz16  that  glucose  behaves  like  a  very  weak  base  in  the 
presence  of  acids,  uniting  with  the  acid  at  the  aldehydic  oxygen 
atom  to  form  an  oxonium  salt.  This  salt,  as  Bunzel  and 
Mathews17  point  out,  dissociates,  though  only  very  slightly,  into 
positive  glucose  ions  C6H1306+  and  the  anion  of  the  respective 
acid.  In  neutral  or  alkaline  solution,  it  was  to  be  assumed  that 
glucose  as  a  weak  acid  would  ionize  into  CeH^Og"  and  hydrogen 
ions.  The  recent  results  of  Mathews  and  Bunzel18  are  in  good 
harmony  with  this  assumption.  The  ionization  process 


was  found  to  be  hastened  considerably  by  adding  OH~  ions,  and 
was  altogether  suppressed  on  adding  a  larger  number  of  H+ 
ions,  whereas  the  process 

C6H1208  +  HC1  =  C6H13(V  +  Cl- 

was  accelerated  to  a  certain  degree  by  acid,  and  very  much 
depressed  in  the  presence  of  alkalies.  We  can  easily  explain 
these  phenomena  on  the  consideration  that  in  both  cases  a  salt 


11  Y.  Osaka,  Die  Birotation  der  d-Glucose,  Zeitschr.  f.  Physik.  Chcmie. 
vol.  35,  p.  661,  1900. 

12  Baeyer  and  Villiger,  Ber.  Deutsch.  Chem.  Ges.,  vol.  34,  pp.  2679,  3612, 
1901;    ibid.,  vol.   35,  pp.   1189,   3013,   1902;    cf.   H.   H.   Bunzel  and   A.   P. 
Mathews,  The  Mechanism  of  the  Oxydation  of  Glucose  by  Bromine  in  Neutral 
and  Acid  Solutions,  Journ.  Am.  Chem.  Soc.,  vol.  31,  p.  464,  1909. 

is  Cohen,  Ber.  Deutsch.  Chem.  Ges.,  vol.  35,  p.  2673,  1902 ;  cf .  Bunzel  and 
Mathews,  loc.  cit. 

i*  Walden,  Ber.  Deutsch.  chem.  Ges.,  vol.  34,  p.  4185,  1901;  cf.  Bunzel 
and  Mathews,  loc.  cit. 

is  Walker,  Ber.  Deutsch.  chem.  Ges.,  vol.  34,  p.  4115,  1901 ;  cf .  Bunzel  and 
Mathews,  loc.  cit. 

iG  J.  Stieglitz,  Studies  in  Catalysis,  Am.  Chem.  Journal,  vol.  39,  pp.  29 
and  166,  1908. 

i^  Loc.  cit. 

is  Loc.  cit;  H.  H.  Bunzel,  The  Mechanism  of  the  Oxydation  of  Glucose  by 
Bromine,  Journ.  Biol.  Chem.,  vol.  7,  p.  157,  1909. 


124        University  of  California  Publications  in  Physiology  [VOL.  4 

formation  must  take  place.  From  the  work  of  Stieglitz19  we 
know  that  salts  of  weak  acids  and  bases  are  ionized  to  a  far 
greater  extent  than  the  respective  acids  or  bases  in  free  state. 
From  the  results  above  it  would  follow  that  we  have  to  regard 
glucose  as  a  true  amphoteric  electrolyte  with  predominating  acid 
character.  The  free  acid  is,  however,  only  very  scarcely  ionized 
and  hence  highly  inactive.  Processes  in  which  the  negative 
glucose  ion  is  involved  will  proceed  with  comparatively  high 
velocity  while  reactions  in  which  the  positive  ion  is  alone  active 
(the  negative  ion  being  suppressed)  proceed  very  slowly. 
Mathews  and  Munzel20  also  tried  to  determine  which  of  these  two 
glucose  ions,  CgH^Og"  and  (C6H130U+)  could  be  oxydized  in  the 
presence  of  bromine  in  different  concentrations  of  alkali  and 
acid,  and  at  what  rate  the  respective  reactions  would  proceed. 
The  progress  of  the  oxydation  was  determined  by  measuring  the 
rate  of  disappearance  of  the  bromine.  Bunzel21  in  his  last 
'publication  arrives  at  the  conclusion  that  in  absolute  neutrality 
the  oxydations  of  both  negative  and  positive  glucose  ions  proceed 
simultaneously,  but  with  different  velocities,  the  oxydation  of 
the  negative  ion  naturally  being  more  rapid  than  the  positive 
ion  oxydation.  In  an  acid  medium  the  rate  of  oxydation  is 
falling  off  in  direct  proportion  to  the  concentration  of  the  acid, 
until  enough  acid  is  present  to  suppress  the  oxydation  of  the 
negative  ion  entirely.  Beyond  this  point  the  addition  of  more 
acid  has  no  depressing  effect.  With  regard  to  the  results  of 
Kuff22  and  others  there  is  strong  evidence  that  gluconic  acid  and 
finally  saccharic  acid  are  the  main  products  of  this  very  slow 
positive  ion  oxydation. 

The  presence  of  alkalies,  in  accordance  with  what  has  already 
been  stated,  greatly  accelerated  the  negative  ion  oxydation  in 
Mathew's  experiments,  until  a  certain  optimal  alkalinity  was 
reached,  beyond  which  the  increment  falls  off  again.  Alkali  is, 
however,  unable  to  suppress  the  negative  ion  oxydation  entirely. 


is  LOG.  cit. 

20  Loc.  cit. 

21  H.  H.  Bunzel  loc.  cit. 

22  O.  Kuff,  Zur  Darstellung  der  einbasischen  Sauren  der  Zuckergruppe, 
Ber.  Deutsch.  chem.  Ges.,  vol.  32,  p.  2273,  1899. 


1912]  Birckner:  Glucose  Oxydations  125 

Mathews23  considers  the  phenomena  depending  on  the 
ionization  (and  the  amphotheric  properties)  of  the  glucose 
molecule24  as  having  considerable  biological  significance.  The 
true  explanation  of  the  rapid  oxydation  of  sugar  in  the  organ- 
ism is,  according  to  him,  not  merely  its  faculty  to  activate  the 
oxygen,  as  had  been  the  almost  general  assumption  for  several 
decades,  but  more  particularly  its  faculty  of  increasing  in  some 
way  or  other  the  "active  mass"  of  the  sugar  itself,  i.e.,  by 
increasing  its  dissociation.  A  simultaneous  activation  of  the 
oxygen  is  of  course  not  impossible,  but  it  is  emphasized  that,  no 
matter  whether  the  sugar  is  to  be  acted  upon  in  an  alkaline  or 
neutral  or  even  in  an  acid  solution,  the  first  prerequisite  of 
oxydation  is  always  an  increased  ionization  of  the  reducing  sub- 
stance, an  activation  of  the  oxygen  possibly  (but  not  necessarily) 
being  effected  at  the  same  time.  Similar  conceptions  of  this 
question  we  find  already  in  the  works  of  Abderhalden25  and 
Schade20.  The  reason  why  glucose  does  not  oxidize  rapidly  in 
vitro  is  to  be  sought  for  chiefly  in  the  glucose  molecule  itself  and 
not  in  the  lack  of  active  oxygen,  and,  applying  this  deduction  to 
well-known  pathological  conditions  of  the  body,  Mathews27 
points  out  "that  a  failure  of  living  matter  to  burn  glucose  is 
probably  not  due  to  the  absence  of  oxydases,  properly  speaking, 
but  to  the  probable  loss  of  its  power  to  dissociate  the  glucose. 
Under  the  term  of  oxydases  there  have  hitherto  been  confused 
two  classes  of  substances;  one  which  activates  the  oxygen;  the 
other,  the  more  important  class,  which  activates  by  dissociation 
the  reducing  substances.  The  latter  are  specific,  the  former  not." 

Although  certainly  of  importance  in  view  of  the  fact  that 
in  nature  sugar  is  being  broken  down  with  apparently  equal 
readiness  in  both  alkaline  and  acid  media,  it  must  not  be  for- 
gotten that  the  dissociation  process  is  only  the  introductory  step 


23  A.  P.  Mathews,  The   Spontaneous  Oxydation  of  the  Sugars,  Journ. 
Biol.  Chem.,  vol.  6,  p.  19,  1909. 

24  Similar  relations  may  with  some  certainty  be  assumed  to  exist   also 
with  regard  to  other  sugars. 

25  E.  Abderhalden,  Lehrbuch  der  Physiologischen  Chemie  (Berlin,  1906), 
p.  102. 

26  H.  Schade,  Die  Bedeutung  der  Katalyse  fur  die  Medicin.  (Kiel,  1907), 
p.  126;  cf.  Lob,  Biochem.  Zeitschrift,  vol.  20,  p.  528,  1909. 

27  Loc.  cit.,  p.  20. 


126        University  of  California  Publications  in  Physiology  [VOL.  4 

for  the  subsequent  chemical  reaction.  The  two  complex  glucose 
ions  must  naturally  have  very  little  or  almost  no  stability,  and 
are  easily  broken  down  into  smaller  groups  of  atoms.  These  in 
turn  may  go  into  more  or  less  stable  combinations,  and  it  can 
a  priori  not  be  anticipated  in  what  way  the  primary  glucose  ions 
may  determine  the  direction  of  these  changes.  We  possess 
altogether  too  little  knowledge  at  present  of  the  nature  of  the 
transformation  products  of  glucose  in  those  solutions,  to  obtain 
much  new  information  from  experiments  of  this  kind.  After 
the  question  of  these  reaction  products,  which  at  present  stands 
in  the  center  of  the  interest,  is  worked  out  more  clearly,  experi- 
ments of  the  type  of  those  to  which  reference  has  just  been 
made  will  undoubtedly  be  valued  much  more  highly. 

3.  The  Action  of  Radiant  Energy  on  Glucose 

(a)   The  Electrolysis  of  Sugars.    Lob's  Theory. 

It  was  not  until  recently  that  the  question  of  the  products 
of  glucose  transformations  has  been  properly  attacked.  The 
simplest  case  is  presented  in  the  experiments  of  Neuberg  on  the 
electrolysis  of  pure  organic  compounds  in  aqueous  solutions. 
Neuberg28  is  the  first  one  to  use  the  pure  substance  without  the 
addition  of  an  electrolyte.  He  subjected  a  large  number  of 
organic  compounds  in  dilute  solution  to  a  direct  current  in  a 
dark  vessel,  using  platinum  electrodes.  The  conductivity  of 
many  of  these  substances,  which  wras  actually  or  nearly  zero  at 
first,  increased  after  they  had  been  exposed  to  the  current  for 
some  time.  With  pure  glucose,  for  instance,  it  took  thirty-five 
minutes  before  the  conductivity  became  measurable.  During  the 
course  of  the  experiment  Neuberg  took  small  samples  from  thex 
liquid  and  made  qualitative  tests,  any  gas  formation  being 
neglected. 

Thus  with  a  2  per  cent  solution  of  glucose  in  pure  distilled 
water,  after  exposing  to  the  current  for  eighteen  hours,  he 
obtained  the  following  reactions: 


28  c.  Neuberg,  Elektrosynthesen  in  der  Zuckerreihe,  Biochem.  Zeitschr., 
vol.  7,  p.  527,  1908;  Chemisehe  Umwandlungen  durch  Strahlenarten  II,  Bio- 
chem. Zeitschr.,  vol.  17,  p.  270,  1909. 


1912]  BircJcner:  Glucose  Oxydations  127 

The  liquid  reduced  Fehling's  solution  almost  instantly 
in  the  cold. 

Likewise  an  alkaline  solution  of  copper  acetate  was 
reduced  after  a  short  time. 

Tollens'29  orcin  reaction  for  pentoses  was  given  but 
very  slightly. 

The  reaction  with  naphtoresorcin30  was  very  strong, 
indicating  the  presence  of  glucuronic  acid  or  of  one  of  its 
homologes. 

On  adding  barium  hydrate,  a  yellow,  flaky  precipitate 
of  a  basic  barium  salt  was  formed. 

With  basic  lead  acetate  the  liquid  gave  a  precipitate 
unlike  the  original  solution.  The  precipitate  dissolved  in 
an  excess  of  the  lead  reagent. 

With  phenylhydrazine  the  liquid  gave  at  once  a  tur- 
bidity in  the  cold,  a  sticky  oil  separating  out  on  standing 
The  filtrate  from  this  oil  soon  gave  crystals  of  glu- 
cosazon  in  the  cold,  the  latter  taking  rise  from  the  d- 
glucosone. 

No  formaldehyde  nor  trioxy-methylene  could  be 
detected. 

The  liquid  was  fermentable  by  yeast. 

Quite  similar  results  were  obtained  with  d-fructose,  cane- 
sugar,  raffinose  and  a-  and  /3-methylglucoside.  The  di-saccharides 
and  tri-saccharides  as  well  as  the  glucosides  apparently  under- 
went first  a  hydrolytic  cleavage,  whereupon  the  glucose  con- 
stituent was  broken  down  in  its  usual  way.  Some  of  the  products 
had  probably  arisen  not  from  the  hexose  directly,  but  through 
secondary  changes  under  the  influence  of  the  current. 

On  the  whole,  Neuberg  could  conclude  from  these  results  that 
the  electrolysis  of  mono-saccharides  even  in  neutral  solution  is 
a  means  of  causing  chemical  transformation  of  these  otherwise 


29  B.  Tollens,  tiber  Farbenreactioneu  auf  Xylose  und  Arabinose,  etc., 
LieUg's  Ann.  d.  Chemie,  vol.  254,  p.  329,  1889. 

so  B.  Tollens,  tiber  einen  einfachen  Nachweis  der  Glucuronsaure  mittels 
Naphtoresorcin,  HC1,  and  Aether,  Ber.  Deutsch.  cliem.  Ges.,  vol.  41,  p.  1788, 
1908. 


128        University  of  California  Publications  in  Physiology  [VOL.  4 

indifferent  substances.  The  main  products  resulting  from  these 
changes  are  carboxylic  acids  and  the  osones  of  the  respective 
sugars. 

"While  Neuberg  carried  out  these  experiments  only  recently, 
Walther  Lob  had  devoted  himself  to  similar  studies  for  a  number 
of  years  previously.  Only  his  last  investigation  on  the  elec- 
trolysis of  grape  sugar  is  of  very  recent  date,  too,  and  it  may  be 
briefly  reviewed  in  connection  with  that  of  Neuberg. 

The  object  of  Lob's  wTork  was  primarily  to  find  whether  the 
simplest  sugar,  HCOH,  could  be  found  among  the  products  of 
electrolysis  of  glucose.31  According  to  some  previous  experi- 
ences, such  a  transformation  seemed  indeed  to  be  possible.  Thus 
Buchner,  Meisenheimer  and  Schade32  had  already  described  the 
formation  of  formic  acid  as  one  of  the  products  of  sugar  oxyda- 
tion  by  hydrogen  peroxide ;  von  Lebedew33  had  observed  f  or- 
maldehyde  among  the  products  of  the  action  of  the  cell-free 
yeast  ferment  on  sugar.  Furthermore,  the  experiments  of 
"Windaus  and  Knoop,34  Neuberg,35  and  Bokorny36  seemed  to 
contain  some  more  or  less  direct  evidence  for  an  intermediary 
formation  of  this  substance.  The  work  of  Walther  Lob37  leaves 
no  doubt  that  formaldehyde  is  actually  one  of  the  regular 
products  of  the  electrolysis  of  grape-sugar.  His  manner  of 
experimentation,  however,  differed  from  the  method  employed 
by  Neuberg  in  several  points.  He  used  his  glucose  in 'a  solution 
containing  5  per  cent  sulphuric  acid,  and  observed  the  changes 


31  For  the  earlier  literature  on  the  electrolysis  of  glucose  see:  E.  von  Lipp- 
mann,  Die  Chemie  der  Zuckerarten  (ed.  3,  Fr.  Vieweg  &  Sohn,  Braunschweig, 
1904),  p.  373;  or  C.  Neuberg,  loc.  cit.,  Biochem.  Zeitschr.,  vol.  17,  p.  270, 
1909. 

32  E.  Buchner,  J.  Meisenheimer  and  H.  Schade,  Alkoholische  Garung  ohne 
Enzyme,  Ber.  Deutsch.  chem.  Ges.,  vol.  39,  p.  4217,  1906. 

33  A.  von  Lebedew,  tiber  das  Auftreten  von  Formaldehyd  bei  der  Zell- 
freien  Garung,  Biochem.  Zeitschr.,  vol.  10,  p.  454,  1908. 

3-t  A.  Windaus  and  F.   Knoop,  Die  uberfiihrung  von   Traubenzucker  in 
Methylimidazol,  Ber.  Deutsch.  chem.  Ges.,  vol.  38,  p.  1166,  1905. 

35  C.  Neuberg,  Depolymerisation  der  Zuckerarten,  Biochem.  Zeitschr.,  vol. 
12,  p.  337,  1908. 

36  Th.  Bokorny,  tiber  die  Assimilation  des  Formaldehyd  und  die  Versuche, 
dieses  Zwichenprodukt  der  Kohlensaure  Assimilation  nachzuweisen,  Pflilger's 
Archiv,  vol.  125,  p.  467,  1908. 

37  W.  Lob,  Zur  Kenntnis   der  Zuckerspaltungen,  m-vii,  Biochem.  Zeit- 
schr., vol.  17,  pp.  132  and  343,  1908;  vol.  20,  p.  516,  1909;  vol.  22,  p.  103, 
1909;  vol.  23,  p.  10,  1909;  Zeitschr.  f.  Elektrochem.,  vol.  16,  p.  1,  1910. 


1912]  Birckner:  Glucose  Oxydations  129 

at  each  pole  separately,  the  one  in  touch  with  the  sugar  always 
being  a  lead  spiral  (cooled  by  water  running  inside),  while  the 
other,  which  was  separated  from  the  former  by  a  diaphragm,  was 
a  platinum  electrode.  The  glucose  was  used  in  a  solution  of  not 
less  than  20  per  cent.  The  potential  of  the  current  was  four  to 
five  volts.  Lob  found  as  the  chief  products  at  either  pole  for- 
maldehyde and  pentose,  showing  at  the  same  time  that  their 
formation  is  due  not  simply  to  the  electrolytic  oxydation  at  the 
anode,  but  takes  place  in  the  same  way,  although  more  slowly, 
under  the  reducing  influence  of  the  kathodic  hydrogen.  At  the 
anode  a  marked  secondary  oxydation  to  the  corresponding  acids 
was  observed.  As  a  consequence  of  the  relatively  high  inactivity 
of  formaldehyde  and  pentose,  their  oxydation  takes  place  far 
more  slowly  than  that  of  the  glucose  itself,  whereby  the  equi- 
librium C6H1206  =  C5H1005  -f  HCOH  is  shifted  towards  the 
right,  resulting  in  an  excess  of  pentose  and  aldehyde.  These  two, 
according  to  Lob,  are  the  real  dissociation  products  of  glucose, 
and  he  assumes  an  equilibrium  of  the  same  type  to  establish  itself 
in  the  aqueous  solution  of  every  aldose  sugar. 

As  already  stated,  he  succeeded  in  showing  that  under  the 
influence  of  the  kathodic  hydrogen  the  equilibrium  was  shifted 
in  the  same  direction  as  at  the  anode,  the  glucose  being  far  more 
readily  reduced  (forming  mannite)  than  its  dissociation  products 
at  the  right  side  of  the  formula. 

Theoretically  there  was,  therefore,  no  intelligible  reason  why 
in  Neuberg's  experiment  no  formaldehyde  reaction  could  be 
obtained.  Lob,38  on  repeating  the  experiment  in  the  manner 
described  by  Neuberg,33  actually  could  make  sure  that  formalde- 
hyde is  also  formed  in  the  electrolysis  of  glucose  in  a  non- 
acidulated  solution  without  the  use  of  a  diaphragm.40 

For  the  reaction  that  takes  place  at  the  lead  anode  in  his 
original  experiment,  Lob41  proposes  the  following  formulation : 


38  Loc.  cit.,  Biochem.  Zeitschr.,  vol.  22,  p.  105,  1909. 

39  Loc.  cit. 

40  For  the  methods  used  by  Lob  for  the  identification  of  formaldehyde 
see  Zeitschr.  f.  Elektrochem.,  vol.  16,  p.  2,  1910. 

41  Loc.  cit. 


130        University  of  California  Publications  in  Physiology  [VOL.  4 


Glucose 
C.Hia0 


Pentose 
C5H1005 


Formaldehyde 
HCOH 


CO,  CO2,  HCOOH 
Formic 
acid 


the  first  line  representing  the  primary  reaction,  the  second  line 
showing  the  products  of  secondary  oxydation  processes. 

Lob  has  shown  in  a  similar  way  that  the  same  relations  do 
also  hold  for  the  transformation  of  the  lower  aldoses  and  their 
respective  alcohols  under  the  influence  of  the  current.  At  least 
the  formation  of  formaldehyde  was  common  to  all  of  them.  The 
formation  of  the  next  lower  aldose,  however,  could  never  be 
detected  so  far  (except  when  starting  from  the  hexoses),  although 
its  initial  formation  is  beyond  doubt.  There  was  noticed  instead, 
however,  a  remarkable  tendency  for  a  re-formation  of  the  more 
stable  pentoses  from  these  lower  and  rather  unstable  depoly- 
merization  products.  Thus,  starting  from  glycerine,  for  instance. 
Lob42  obtained 

( 1 )  a  fair  amount  of  formaldehyde ; 

(2)  a   syrupy   mass,    free   from   glycerose,    glycolose, 
dioxyaceton     and     hexose,     but     containing     a     pentose 
(i-arabinose). 

Lob  regards  as  the  introductory  step  of  this  reaction  the 
formation  of  glycerose  by  the  anodic  oxygen  (Glycerine  -{-  0  = 
glycerose  -f  H20),  which  sugar  at  once  dissociates  into  glycolose 
and  formaldehyde,  the  glycolose  and  glycerose  in  statu  nascendi 
uniting  to  form  pentose. 

In  analogy  to  the  formulation  for  glucose,  these  changes  may 
be  represented  as  follows: 


42  Loc.  cit.,  Zeitschr.  f.  EleJctrochem.,  vol.  16,  p.  5,  1910 ;  Biochem.  Zeit- 
schr.,  vol.  17,  p.  343,  1909. 


1912] 


Birckner:  Glucose  Oxydations 


131 


Glycerose 
C3H003 


Glycolose 
C2H4O2 


Formaldehyde 
CH20 


HCOOH,  CO,  CO2 


C5HS07 

Trioxy-glutaric 
acid 


Through  these  experiments,  Lob  has  not  only  illustrated  a 
new  way  by  which  hexoses  may  be  changed  into  pentoses,  but  he 
has  also  established  the  fact  that  the  synthesis  of  glucose  is  a 
reversible  process  in  strictly  chemical  sense,  and  that  we  have 
a  right  to  speak  of  a  depolymerization  of  the  hexose  molecule. 
By  means  of  electrolytic  methods,  this  molecule  can  be  caused  to 
give  off  gradually  all  those  six  formaldehyde  groups  from  which 
it  was  originally  built  up. 

It  should  be  stated  here  that  the  experiments  just  referred  to 
furnished  only  a  confirmation  of  the  remarkable  theory  that 
Walther  Lob  had  already  worked  out  previously  from  results 
of  others  and  his  own.  I  consider  it  worth  while  to  say  a  few 
words  about  these  theoretical  deductions  of  an  author  whose 
work  has  contributed  so  much  to  the  better  understanding  of  the 
mechanics  of  sugar  synthesis  and  transformations.  His  theory, 
fantastic  as  it  may  have  impressed  the  reader  at  first,  enables  us 
to  look  upon  these  problems  from  quite  a  new  standpoint,  .and 
may  possibly  contain  the  key  to  important  discoveries  in  the 
future. 

The  fundamental  basis  of  Lob's  theory  is  the  well-known 
hypothesis  of  Baeyer,43  according  to  wrhich  the  assimilation  of 
carbon  dioxyde  through  the  green  parts  of  the  plant  takes  place 
essentially  in  two  steps.  The  C02  is  first  reduced  by  the  action 
of  the  chlorophyll  to  formaldehyde,  and  the  latter  then  condenses 


43  A.  von  Baeyer,  Tiber  die  Wasserentziehung  und  ihre  Bedeutung  f  iir 
das  Pflanzenleben  und  die  Canning,  Ber.  Deutsch.  chem.  Ges.,  vol.  3,  p.  63, 
1870. 


132        University  of  California  Publications  in  Physiology  [VOL.  4 

to  form  sugar.     The  process  is  frequently  expressed  by  the  fol- 
lowing two  chemical  equations : 

CO2  +  Ha  =  H  COOH  +  Oa  (1) 

6  HCOH  =  C6H12O6  (2) 

This  hypothesis  has  withstood  the  attacks  of  four  decades, 
and  according  to  the  present  views,  we  possess  no  other  theory 
that  explains  the  facts  observed  so  well  as  this.  It  has  lately 
been  confirmed,  especially  by  the  results  of  Losanitsch,44  Lob,45 
Fenton,46  and  Usher  and  Priestly.47 

As  the  correctness  of  this  theory  became  more  and  more 
obvious,  W.  Lob,48  extending  it  to  the  reversed  reaction,  con- 
cluded that  formaldehyde  is  really  the  central  figure  in  the 
following  three  biochemical  processes 

( 1 )  the  synthesis  of  sugar  in  the  plant ; 

(2)  the  respiratory  oxydation; 

(3)  the  alcoholic  fermentation. 

Artificial  syntheses  of  glucose  by  purely  chemical  means,49  as 
well  as  those  by  silent  electric  discharge,30  had  shown  that 
formaldehyde  is  among  the  intermediary  products. 

Now  formaldehyde  on  account  of  its  highly  toxic  qualities 
cannot  exist  in  the  tissues  as  such.  It  is,  furthermore,  in  its 
usual  state  too  inactive  to  combine  readily  with  other  radicles. 
Lob  therefore  assumes  it  to  be  present  in  a  "tautomeric,  active," 


44  S.  M.  Losanitsch,  tiber  die  Elektroynthesen,  Ber.  Deutsch.  diem.  Ges., 
vol.  40,  p.  4656,  1907. 

45  W.  Lob,  Zur  Kenntnis  tier  Assimilation  cler  Kohlensaure,  Zeitschr.  f. 
Elektrochem.,  vol.   11,   p.   745,   1905;    Lanclw.  Jahrbiicher,   vol.   35,   p.   541, 
1906;    Studien    iiber    die    chemischen    Wirkungen    der    stillen    elektrischen 
Entladung,  Zeitschr.  f.  Elektrochem.,  vol.  12,  p.  282,  1906. 

46  H.  J.  Fenton,  The  Eeduction  of  Carbon  Dioxide  to  Formaldehyde  in 
Aqueous  Solutions.    Journ.  Chem.  Soc.,  vol.  91,  p.  687,  1907. 

47  F.  L.  Usher  and  J.  H.  Priestley,  The  Mechanism  of  Carbon  Assimila- 
tion in  Green  Plants,  Proc.  Boy.  Soc.   (B),  vol.  77,  p.  369,  1906;  ibid.,  vol. 
78,  p.  318,  1906;  ibid.,  vol.  84,  p.  101,  1911. 

48  W.  Lob,  Zur  chemischen  Theorie  der  Alkoholgarung,  Zeitschr.  f.  Elek- 
trochem., vol.   13,  p.  511,   1907;   Zur  Geschichte  der  chemischen  Gahrungs- 
hypothesen,  Biochem.  Zeitschr.,  vol.  29,   p.  311,   1910,  and   literature   cited 
on  page  — •  of  this  article. 

49  Butlerow,  E.  Fischer,  O.  Low,   Tollens;    cf.   Neuberg,  Biochem.  Zeit- 
schr., vol.  24,  p.  152. 

so  Berthelot,  Thenard,  Brodie,  Maquenne,  Losanitsch  und  Jovitschitsch, 
Butlerow,  W.  Lob;  cf.  Neuberg,  loc.  cit. 


1912J  Birckner:  Glucose  Oxydations  133 

form  in  which  it  cannot  be  detected  by  the  usual  reactions.  By 
submitting  a  mixture  of  alcohol  and  C02  in  alkaline  solution  to 
the  silent  electric  discharge,  Lob  also  succeeded  in  proving  the 
intermediate  formation  of  CO  and  H2  during  this  slow  synthetic 
process.  As  the  simplest  way  of  representing  his  active  tauto- 
meric  modification  of  formaldehyde,  I  would  hence  suggest  the 
following  equilibrium, 

COH2=(CO  +  H2), 

the  unstable  form,  represented  by  the  right  side,  being  almost 
alone  present  in  the  tissues  and  in  reactive  mixtures. 

Only  if  under  certain  conditions  (for  instance,  in  those 
artificial  syntheses)  the  rate  of  transformation  becomes  abnorm- 
ally slow,  these  unstable  compounds  to  the  right  will  accumulate 
in  such  quantities  as  to  cause  a  shifting  of  the  equilibrium 
towards  the  left,  i.e.,  a  formation  of  the  stable  compound.  We 
may  therefore  regard  this  unstable  combination  of  a  CO  and 
a  H2  group  as  the  "active  mass"  of  the  formaldehyde,  the  for- 
mation of  which  must  precede  every  reaction  in  which  formalde- 
hyde is  actually  involved,  no  matter  whether  a  higher  aldehyde 
is  being  broken  down  to  C02  and  water,  or  whether  it  is  being 
synthesized  from  them.  In  both  cases  the  final  products  are 
ultimately  resulting  from  some  intramolecular  rearrangement, 
either  by  mutual  oxydation  or  reduction,  between  the  CO  and  H 
components  of  the  "active"  formaldehyde  molecules.  Accord- 
ingly, Lob  represents  the  synthesis  of  sugar  and  its  reversed 
processes  in  the  following  manner : 

Sj-nthetic  process. 

6  CO  +  6  H2  =  C6H12O6  endotliermic  (intramol.  reduction) 

Fermentative  process. 

6  CO  +  6  H2  =2  C.H.OH  +  2  CO,  1    exotll€rmic 

Oxydative  process.  >  (intramol.  oxyda- 

6  CO  +  6  H2  +  6  O2  =  6  CO3  +  6  H,O  tion) . 

The  essential  course,  for  instance,  of  the  alcoholic  fermenta- 
tion, according  to  this  scheme,  would  be  the  following: 

The  sugar  under  the  influence  of  zymase  dissociates  com- 
pletely into  active  (tautomeric)  formaldehyde  groups,  the  active 


134        University  of  California  Publications  in  Physiology  [VOL.  4 

components  of  which  recombine  by  a  sort  of  intramolecular 
oxydation  (eliberating  heat)  to  form  alcohol  and  carbon  dioxyde. 

The  first  part  of  the  process  (complete  depolymerization  of 
the  sugar  molecule  into  active  (tautomeric)  formaldehyde 
groups)  is  the  common  precursor  of  all  three  reactions  quoted 
above.  It  is  only  in  the  second  stage  (synthetic  phase)  that 
differences  occur  depending  on  the  respective  experimental  con- 
ditions (the  presence  or  absence  of  oxygen,  etc.).  The  unstable 
groups  CO  and  H2,  it  is  easily  seen,  may  indeed  form  a  great 
variety  of  combinations  the  nature  of  which  will  be  determined 
solely  by  the  conditions  and  the  relative  stability  of  the  respective 
compounds. 

The  synthesis  of  sugar  over  formaldehyde,  either  artificially 
or  in  nature,  is  therefore  in  its  main  part  only  a  special  one  out 
of  a  number  of  coordinate  reactions,  and  the  same  would  be  true 
for  the  synthetic  formation  of  pentoses  from  glycerine,  which 
process  was  mentioned  above. 

The  term  "dissociation  products"  of  glucose,  which  has  been 
used  several  times  in  the  text  of  this  section,  is  perhaps  apt  to 
cause  some  confusion.  Lob  in  fact  assumes  the  existence  of  an 
equilibrium  in  aqueous  solution  between  glucose  and  its  dis- 
sociation products,  which  as  such  would  not  need  to  have  the 
properties  of  true  ions/'1  At  least  one  of  the  dissociation 
products  must  be  the  unstable  form  of  formaldehyde  (CO.H2). 
The  other  dissociation  product  may  be  a  pentose  or  some  lower 
aldose  or  ketose.52  Under  certain  conditions  glucose  may  be  com- 
pletely dissolved  into  (CO.H2)  groups.  Now  supposing  we  have 
the  system 

C,H120.=  (CO.H2)  +  CSH1003. 

•». 
It  is  not  to  be  understood  that  this  equilibrium  can  be  traced 

chemically.  It  is  in  fact  under  normal  conditions  so  much  in 
favor  of  the  hexose,  that  no  formaldehyde  or  pentose  tests  will 


51  Similar  assumptions  are  also   met   with   in   the   paper   of  J.   U.   Nef, 
Dissociationsvorgange   in   der  Zuckergruppe   I,   Liebig's  Ann.   d.   Chcmie, 
vol.  357,  p.  214,  1907. 

52  No  distinction  needs  to  be  made  here  between  aldose  and  ketose  sugars 
since  they  are  easily  convertible  into  one  another,  as  we  know  from  the  work 
of  Lobry  de  Bruyn,  Ber.  Deutscli.  chem.  Ges.,  vol.  28,  p.  3078,  1895,  and  of 
Wohl  and  Neuberg,  ibid.,  vol.  33,  p.  3099,  1900. 


1912]  Birckner:  Glucose  Oxydations  135 

be  given.  Nor  would  it  be  possible  by  arranging  conditions  in 
such  a  manner  as  to  induce  a  synthetic  formation  of  sugar  from 
formaldehyde,  to  obtain  any  more  hexose.  Both  dissociation 
products  are  hence  to  be  assumed  as  existing  in  some  unstable 
(potentially  active)  state,  and  yet  we  are  able  to  create  con- 
ditions53 under  which  these  labil  dissociation  products  become 
concentrated  enough  to  condense  partly  into  their  stable  form, 
whereupon  they  become  detectable  by  chemical  means.  This 
depolymerization  process,  which  helps  to  loosen  the  molecule  and 
which  is  the  prerequisite  of  any  sugar  oxydation,  may  possibly 
be  brought  about  in  the  living  body  by  some  ferment  acting  on 
the  sugar;  at  any  rate,  Lob,  like  Mathews  (loc.  cit.)  ascribes  an 
inability  of  the  organism  to  burn  sugar  to  conditions,  in  the 
body,  which  are  very  unfavorable  to  the  dissociation  process. 

This  unique  and  very  original  theory  of  Lob  does  not  rest 
merely  on  speculative  grounds,  but  is  the  result  of  many  years 
of  experimental  work.  One  more  instance  may  follow : 

Lob54  boiled  a  solution  of  pure  formaldehyde  with  zinc  dust, 
using  a  reflux  condensor.  From  the  products  obtained  (chiefly 
polyoxy-acids,  volatile  ketones,  especially  methylketol  and  acetol, 
and  a  syrupy  sugar  residue)  it  was  certain  that  the  first  stage 
of  the  reaction  had  been  a  synthetic  formation  of  sugar.  On 
repeating  the  experiment  with  glucose  instead  of  formaldehyde, 
the  products  were  exactly  the  same,  only  in  different  quantitative 
proportions  (as  was  to  be  expected).  Lob  thus  obtained  evidence 
of  the  fact  that  the  decomposition  of  boiling  glucose  under  the 
catalytic  influence  of  zinc  forms  only  a  part  of  the  reaction  that 
takes  place  with  formaldehyde  under  the  same  conditions. 

Summing  up  the  main  results  of  Lob 's  work  we  may  say : 

(1)  The  oxydative  disintegration  of  the  sugar  mole- 
cule, under  all  circumstances,  takes  place  in  two  different 
stages : 

(a)  A  depolymerization  process    (splitting  off  of 

one  or  more  tautomeric  (CO.H2)  groups). 


53  See  the  experiments  of  Neuberg  and  Lob  cited  in  the  first  part  of  this 
section. 

54  Biochem.  Zeitschr.,  vol.  12,  p.  466. 


136        University  of  California  Publications  in  Physiology  [VOL.  4 

(6)   An  oxydation  process   (synthesis  of  C02  and 
H2O  or  of  C2HDOH  and  C02). 

The  alcoholic  fermentation  is  therefore  essentially 
a  synthetic  process. 

(2)  The  two  stages  enumerated  under  (1)  a  and  b, 
represent  not  only  the  biological  but  the  strictly  chemical 
reversion  of  the  process  of  sugar  synthesis. 

I  may  call  attention  to  the  notable  fact  that  this  theory  is  at 
the  same  time  an  excellent  illustration  of  the  close  relationship 
existing  between  normal  respiration  and  alcoholic  fermentation, 
which  was  long  predicted  by  Pfeffer.55 
(b)   The  Action  of  Light. 

Interesting  experiments  have  been  carried  out  lately  by 
Neuberg  and  P.  Mayer  on  the  effect  of  light  on  solutions  of 
glucose. 

The  biological  importance  of  both  light  rays  and  electric  rays 
has  long  been  known  to  medicine,  and  both  forms  are  widely  used 
in  therapeutics,  especially  of  recent  years.  Almost  nothing  was 
known,  however,  about  the  chemical  function  of  these  agents 
until  quite  recently. 

Only  few  organic  substances,  indeed,  are  sensitive  enough  to 
undergo  spontaneous  changes  under  the  influence  of  light,  with 
a  measurable  velocity.  Catalytic  substances  must  therefore  be 
employed  with  necessity  in  the  study  of  these  phenomena. 

Neuberg56  found  the  salts  of  uranium  to  be  very  apt  for  this 
purpose.  He  investigated  the  influence  of  light  on  many  organic 
substances  in  1  to  5  per  cent  solutions,  wrhich  contained  from 
0.5  to  1  per  cent  of  uranium  salt.  Most  of  the  samples,  which 
contained  the  catalysor,  showed  changes  within  a  short  time  (a 
few  hours  or  even  minutes)  after  being  exposed  to  the  sun-rays, 
while  the  control  samples  in  the  dark-room  remained  unchanged. 
Corresponding  sets,  which  had  been  exposed  to  the  light  with- 
out the  addition  of  the  catalysor,  remained  likewise  unchanged. 
The  reactions  which  took  place  were  mostly  of  an  oxydative 


55  W.  Pfeffer,  Pflanzenphysiologie,  vol.  1,  p.  555,  1897.  See  also  Czapek, 
Die  Atmung  d.  Pflanzen,  Ergebn.  d.  Physiol.,  vol.  9,  p.  605,  1910. 

50  C.  Neuberg,  Chemische  Umwandlungen  durch  Strahlenarten,  I,  Bio- 
chem.  Zeitschr.,  vol.  13,  p.  305,  1908. 


Birckner:  Glucose  Oxydations  137 

character,  the  hexoses  generally  being  transformed  to  the  respec- 
tive osones,  cZ-glucose  also  giving  a  positive  pentose  reaction 
with  orcin.57  It  could  also  be  observed  that  the  amount  of  change 
varied  greatly  with  the  intensity  of  the  light,  and  with  light 
from  different  sources.  That  a  possible  contamination  of  the 
uranium  salts  with  traces  of  radium  had  nothing  to  do  with 
their  catalytic  action  was  shown  in  the  first  place  by  the  fact 
that  the  samples  in  the  dark-room  underwent  no  change. 
.  Furthermore,  Neuberg  ascertained  in  special  experiments  that 
even  strong  preparations  of  radium  were  without  influence  on 
his  substances. 

In  a  later  communication,  Neuberg58  describes  similar  experi- 
ments with  iron  salts,  which  led  essentially  to  the  same  results. 

P.  Mayer59  has  very  recently  studied  the  destruction  of 
glucose  by  light  under  the  catalytic  influences  of  traces  of  alkali. 
He  measured  the  progress  of  the  transformation  by  the  changes 
in  optical  rotation  and  in  reducing  power.  These  transforma- 
tions were  not  the  same  as  those  caused  by  alkali  alone  in  higher 
concentration  (in  which  case  acids  are  the  main  products),  but 
under  the  influence  of  the  rays  of  a  Heraeus  quartz  lamp,  Mayer 
observed  largely  the  same  products  that  Neuberg  had  foun4  in 
the  experiments  to  which  reference  has  just  been  made. 

On  the  whole,  it  follows  clearly  from  all  these  investigations 
that  by  using  certain  simple  catalytic  agents,  many  substances 
which  as  such  have  no  photochemical  qualities,  and  among  which 
are  the  common  sugars,  can  undergo  rapid  transformations 
when  exposed  to  the  light.  These  changes  are  a  specific  function 
of  the  light,  and,  as  both  observers  have  found,  they  are  inde- 
pendent, within  wide  limits,  of  variations  of  temperature.  No 
exact  quantitative  data  are,  however,  available  at  present  as  to 
the  extent  of  these  transformations  nor  concerning  the  influence 
of  different  factors. 

We  know  that  owing  to  its  structure  the  plant  is  especially 
susceptible  to  light-actions.  We  further  know  that  electric  forces 


57  Tollens,  loc.  tit. 

58  C.  Neuberg,  Chem.  Umwandl.  durch  Strahlenarten,,  iv,  Biochem.  Zeit- 
schr.,  vol.  29,  p.  379,  1910;  v,  ibid.,  vol.  39,  p.  158,  1912. 

59  P.  Mayer,  tiber  Zerstornng  des  Traubenzuckers  dureh  Lieht,  Biochem. 
Zeitschr.,  vol.  32,  p.  1,  1911. 


138        University  of  California  Publications  in  Physiology  [VOL.  4 

are  active  in  the  animal  body.  We  may  finally  take  it  for 
granted  that  the  catalytic  complements  of  both  forms  of  radiant 
energy  are  normally  present  in  living  matter.  Hence  it  is 
obvious  that  light  rays  as  well  as  electric  potentials  may  be 
reckoned  as  being  among  the  factors  by  which  in  part  the 
oxydative  disintegration  of  organic  matter  in  the  body  may  be 
brought  about. 

4.  The  Action  of  Alkalies  and  Acids  on  Glucose 

That  sugars  may  be  easily  decomposed  by  alkalies60  is  a  very 
old  experience.  It  is,  however,  only  very  recently  that  one  has 
studied  the  nature  of  these  processes  more  closely.  Thus,  the 
knowledge  that  these  changes  can  be  effected  by  the  alkali  as 
such,  i.e.,  by  the  hydroxyl  ion,  is  of  rather  new  date.  Meisen- 
heimer61  was  the  first  to  show  conclusively  that  glucose  can  be 
decomposed  by  dilute  NaOH  with  the  formation  of  acids,  also 
in  the  absence  of  oxygen.  The  hydroxyl  ion,  therefore,  not  only 
hastens  the  dissociation  process,  but  it  causes  the  indifferent 
molecule  to  break  down  into  more  reactive  bodies  by  some  sort 
of  intramolecular  rearrangement.  This  fact  at  the  same  time 
explains  readily  why  Mathews62  observed  far  more  rapid  oxyda- 
tions  with  alkaline  glucose  solutions,  which  had  previously  been 
kept  for  some  time  in  an  atmosphere  of  hydrogen,  than  with 
those  that  had  not  been  treated  that  way.  Mathews  also  observed 
that  normally  in  the  presence  of  air  the  rate  of  oxydation  was 
accelerated  by  alkali  but  only  up  to  a  certain  optimal  alkalinity 
(between  n  and  2n  NaOH),  beyond  which  the  velocity  of  oxyda- 
tion falls  off  again.  MathewTs  ascribes  this  phenomenon  mainly 
to  a  hindering  effect  that  the  strong  alkali  apparently  exerts  on 
the  rate  of  solution  or  on  the  chemical  action  of  the  oxygen. 


oo  The  mechanism  of  the  interconversion  of  glucose  into  mannose  and 
fructose  under  the  influence  of  alkali,  which  phenomenon  was  first 
observed  by  Lobry  de  Bruyn  and  Van  Ekenstein,  is  not  very  well  under- 
stood; and  as  this  reaction  does  not  involve  a  permanent  rupture  of  the 
molecule  or  yield  a  stable  oxydation  product,  its  discussion  may  be 
omitted  in  this  abstract.  (For  reference  see:  Eec.  trav.  chim.  d.  Pays-Bus, 
vol.  14,  pp.  156  and  203,  1895.) 

61  J.  Meisenheimer,  tiber  das  Verhalten  von  Glucose,  Fructose  u.  Galac- 
tose  gegeniiber  verdiinnter  Natronlauge,  Ber.  Deutsch.  chem.  Ges.,  vol.  41, 
p.  1009,  1908. 

62  A.  P.  Mathews,  Journ.  Biol.  Chem.,  vol.  6,  p.  3. 


1912]  Birckner:  Glucose  Oxydations  139 

A  considerable  amount  of  study  has  been  devoted  to  the 
question  whether  or  not  an  alkalinity  corresponding  to  that  of 
our  blood,  when  acting  on  glucose  at  body  temperature,  could 
bring  about  the  destruction  of  the  sugar  with  some  rapidity.  Up 
to  very  recently  this  was  indeed  generally  supposed  to  be  the 
case,  as  with  the  old  titration  methods  the  alkalinity  of  the 
blood  was  found  much  higher  than  it  actually  is,  if  only  the 
concentration  of  the  OH"  ions  is  taken  into  account.  For  pre- 
paring solutions  of  very  low  alkalinity  the  use  of  free  alkalies  is 
not  yery  suitable;  but  mixtures  of  monobasic  and  di-basic 
phosphates  have  been  used  with  much  advantage.  By  using  such 
mixtures  Michaelis  and  Rona63  have  shown  not  long  ago  that  an 
alkalinity  equal  to  that  of  human  blood64  causes  no  measurable 
destruction  of  glucose  in  dilute  solution  in  one  day. 

It  would  be  unjustified,  however,  to  apply  this  result,  the 
correctness  of  which  as  it  stands  can  not  be  doubted,  to  physio- 
logical conditions  without  certain  restrictions,  as  has  really  some- 
times been  done.05  As  W.  Lob66  rightly  points  out,  it  is  very 
well  possible  that  even  the  slight  alkalinity  of  the  blood,  which 
as  such  would  be  practically  without  action  on  the  sugar,  might 
be  very  much  accelerated  in  its  action  by  other  substances  in 
the  blood  which  may  act  as  catalysors.  This  point  has  been 
overlooked  by  Michaelis  and  Rona. 

Indeed,  their  results  become  very  different  if  the  solutions  be 
supplied  with  an  ample  amount  of  active  oxygen,  either  by 
bubbling  a  current  of  oxygen  gas  through  the  liquid  or  by 
adding  some  H202.  It  is  well  known  that  active  oxygen  as  well 
as  oxydizing  substances  are  always  present  in  the  blood. 

AVith  an  alkalinity  of  less  than  N/10  NaOH  in  dilute  glucose 
solution,  and  in  the  presence  of  an  oxydizing  agent  Lob67  not 
only  observed  changes  in  the  sugar  content,  but  was  able  after 
twenty  hours  to  determine  quantitatively  the  amount  of  formic 


03  L.  Michaelis  and  P.  Bona,  Die  Alkaliempfindlichkeit  des  Trauben- 
zuckers,  Biocliem.  Zeitschr.,  vol.  23,  p.  364,  1909. 

0-4  The  concentration  of  H+  ions  in  blood  is  0.3  X  10~7;  that  of  water 
at  blood  temperature,  0.85  X  10~T. 

65  See  P.  Mayer,  loc.  cit.,  p.  2. 

oo  W.  Lob,  Zur  Frage  der  Glycolyse,  I,  Biochem.  Zeitschr.,  vol.  29,  p. 
316,  1910. 

07  Biochem.  Zeitschr.,  vol.  23,  p.  22,  1909. 


140        University  of  California  Publications  in  Physiology  [VOL.  4 

acid  formed,  and  besides  to  obtain  qualitative  tests  for  formalde- 
hyde and  pentose.  That  changes  occur  under  these  conditions  of 
very  low  alkalinity,  is  also  affirmed  by  the  extensive  studies  of 
Jolles68  on  the  destruction  of  the  different  sugars  at  body  tem- 
perature. Jolles  kept  all  his  solutions  at  a  constant  alkalinity 
of  N/10  NaOH,  by  adding  fresh  alakli  in  proportion  as  the 
original  amount  became  neutralized.  The  accelerating  influence 
on  the  acid  formation  of  the  addition  of  hydrogen  peroxyde  was 
readily  observed.  But  while  according  to  Lob's  last  communi- 
cation69 oxydizing  agents  in  neutral  solutions  are  without  effect 
at  ordinary  temperature,  Jolles  70  reports  quite  recently  a  very 
different  result. 

In  a  neutral  2  per  cent  solution  of  glucose,  to  which  only 
hydrogen  peroxyde  had  been  added,  he  was  able  to  prove  the 
formation  of  glucuronic  acid  after  six  days  at  a  temperature  of 
37°  C.  in  two  different  cases. 

Jolles  also  made  complete  analyses  of  his  alkaline  sugar 
solutions.  Thus,  for  instance,  he  found  that  an  n/100  alkaline 
NaOH)  solution  of  pure  dextrose  (3  per  cent),  on  standing  at 
37°  C.  for  almost  five  months  without  the  addition  of  an  oxydiz- 
ing agent,  contained  at  the  end  of  this  time  ethyl  alcohol,  formic 
acid,  acetic  acid,  and  lactic  acid.  Unfortunately  he  does  not 
make  any  statement  as  to  how  the  solution  was  kept  sterile  for 
such  a  long  time,  a  fact  which  would  of  course  be  important  to 
know  for  the  correct  interpretation  of  these  results. 

With  regard  to  the  influence  of  weakly  alkaline  phosphate 
mixtures  on  glucose,  W.  Lob  in  his  last  paper,  which  I  just  took 
occasion  to  refer  to,  finds  a  new  fact  of  importance,  namely  that 
in  the  presence  of  H20.>  the  phosphate  ion  P0~4  itself  has  a  dis- 
tinctly catalytic  influence  on  the  action  of  the  OH  ions  on 
glucose,  which  influence  is  still  perceptible  if  the  hydrogen  ion 
concentration  of  the  liquid  becomes  greater  than  that  of  water, 
i.e.,  if  the  fluid  alreday  shows  an  acid  reaction.  This  influence 
of  the  P04  ion  is  specific  to  this  group,  and  increases  in  direct 


os  A.  Jolles,  Zur  Kenntnis  des  Zerfalls  der  Zuckerarten,  Biochem.  Zeit- 
schr., vol.  29,  p.  152,  1910. 

ca  Biochem.  Zeitschr.,  vol.  32,  p.  47,  1911. 

"o  A.  Jolles,  tiber  eine  neue  Bildungsweise  der  Glucuronsaure,  Biochem. 
Zeitschr.,  vol.  34,  p.  242,  1911. 


1912]  Birckner:  Glucose  Oxydations  141 

proportion  with  its  concentration,  the  concentration  of  the  OH" 
ions  being  kept  constant.  This  catalytic  effect  of  the  phosphate 
ion  can  be  greatly  depressed  or  even  inhibited  by  the  addition 
of  organic  derivatives,  especially  of  peptons,  proteins,  or  sera. 

Of  other  substances  that  show  an  action  on  glucose  by  means 
of  OH~  ions  which  they  form  in  contact  with  water,  those  that 
have  been  particularly  studied  are  the  metals,  zinc,  lead  (Lob, 
loc.  cit.}  and  potassium.71 

The  formation  of  the  nitrogen  compound  methyl-glyoxaline 
from  glucose  by  the  action  of  zinc  hydroxyde  ammonia,  at 
ordinary  temperature,  as  observed  by  Windaus  and  Knoop,72 
may  also  be  mentioned  in  this  connection. 

In  turning  to  the  question  as  to  what  are  the  products  of  the 
action  of  alkali  on  glucose,  we  have  to  distinguish  between  the 
specific  effect  of  the  OH"  group  as  such,  and  its  action  as  catalyst 
in  sugar  oxydations. 

The  product  characteristic  of  the  action  of  OH"  groups  on 
glucose  in  the  absence  of  air  is  lactic  acid.  Buchner,  Meisen- 
heimer  and  Schade73  from  a  2  per  cent  solution  of  glucose  in 
n/NaOH  in  closed  vessels  recovered  as  much  as  50-60  per  cent 
of  the  sugar  in  form  of  lactic  acid74  after  prolonged  standing  at 
low  temperature.  This  transformation  represents  the  chemical 
analogon  of  the  lactic  acid  fermentation. 

Nef73  in  his  laborious  studies  found  that  under  the  influence 
of  NaOH7G  the  main  products  of  sugar  decomposition  besides  the 
racemic  form  of  lactic  acid  are  a  mixture  of  saccharins  CGH1005, 


71  J.  Stoklasa,  tiber  die  Zuckerabbau  fordernde  Wirkung  des  Kaliums, 
etc.,  Zeitsclir.  f.  Physiol.  Chemie.,  vol.  62,  p.  47,  1909. 

72  A.  Windaus  and  F.  Knoop,  three  articles  in  Ber.  d.  Deutsch.  chem. 
Ges.,  vol.  38,  p.  1166;  vol.  39,  p.  3886;  vol.  40,  p.  799. 

73  E.   Buchner,   J.   Meisenheimer   and   H.    Schade,   Zur   Vergarung   des 
Zuckers  ohne  Enzyme,  Ber.  Deutsch.  chem.  Ges.,  vol.  39,  p.  4217,  1906. 

7  4  The  formation  of  lactic  acid  from  dextrose  by  the  action  of  alkali 
had  previously  been  observed  by: 

F.  Hoppe-Seyler,  Ber.  Deutsch.  chem.  Ges.,  vol.  4,  p.  346,  1871. 
Kiliani,  ibid.,  vol.  15,  p.  701,  1882. 
Schiitzenberger,  Compt.  rend.,  vol.  76,  p.  440,  1873. 
Framm,  Pfliiger's  Archiv.,  vol.  64,  p.  575,  1896. 

75  J.  U.  Nef,  Dissociationsvorgange  in  der  Zuckergruppe,  Liebig  's  Ami. 
d.  Chem.,  vol.  357,  p.  214,  1907;  ibid.,  vol.  376,  p.  1,  1910. 

76  Not  only  of  Ca(OH)o  (Kiliani,  Ber.  Deutsch.  chem.  Ges.,  vol.  15,  p. 
2960;  vol.  26,  p.  1650;  vol.  35,  p.  3530;  cf.  Nef,  loc.  cit.  I,  p.  303. 


142        University  of  California  Publications  in  Physiology  [VOL.  4 

a  statement  the  correctness  of  which  was  at  first  doubted  by 
Meisenheimer.77  Nef  also  found  that  the  variety  of  products  is 
much  smaller  after  the  action  of  strong  (8n)  NaOH  than  with 
lower  alkalinities,  in  which  latter  cases  it  becomes  extremely 
difficult  to  separate  and  define  the  multitude  of  products. 

If  oxydizing  agents  such  as  metallic  oxydes  are  employed 
together  with  the  alkali,  no  saccharin  formation  takes  place 
(Nef,  loc.  cit.}.  The  products  in  this  case  are  largely  polyoxy- 
acids  and  aldehydes. 

As  is  generally  known,  the  oxydation  of  sugars  by  metallic 
oxydes  in  alkaline  solutions  forms  the  principle  of  many  methods 
of  sugar  determinations.  The  oxydizing  agent  most  commonly 
used  is  known  under  the  name  of  Fehling's  solution,  a  strongly 
alkaline  liquid  containing  cupric  salt  and  potassium-sodium 
tartrate.  In  the  presence  of  aldoses  or  ketoses  the  cupric  salt 
on  heating  is  reduced  to  cuprous  salt,  the  amount  of  which  can 
be  determined  either  by  volumetric  or  by  gravimetric  methods. 

It  should  be  stated  that  all  methods  based  on  this  principle 
are  purely  empirical.  The  course  of  the  reaction  is  unknown, 
and  it  has  no  definite  endpoint.  The  investigator,  therefore,  is 
compelled  to  adhere  rigidly  to  the  directions  given  to  him  by 
the  man  who  worked  out  the  respective  sugar  tables.  About 
forty  differently  composed  Fehling's  solutions  have  so  far  been 
devised.  The  modifications  which  have  been  found  to  give  the 
most  satisfactory  results,  and  which  are  the  ones  most  widely 
used  at  present  are  those  of  Allihn,78  Pfliiger,70  Bertrand,80  and 
I.  Bang.81  In  connection  with  Pfliiger 's  method,  which  is  fre- 
quently used  in  medical  laboratories  for  the  determination  of 


77  J.  Meisenheimer,  loc.  cit. 

78  Allihn,    tiber    den    Verzuckerungsprocess    bei    der    Einwirkung    von 
verdiinnter   Schwefelsaure  auf   Starkemehl  bei  hoherer   Temperatur,  Journ. 
f.  prakt.  Chemie,  N.F.,  vol.  22,  p.   46,   1880.     For  two   other  articles   see 
v.  Lippmann,  Chemie  der  Zuckerarten  (Ed.  3;  Braunschweig,  Fr.  Vieweg 
und  Sohn,  1904),  p.  591. 

79  E.  Pfliiger,  tiber  eine  neue  Methode  zur   quantit.   Bestimmung   des 
Zuckers,  etc.,  Pfliiger 's  Arch.  f.  d.  ges.  Physiol.,  vol.   66,  p.   635.     Unter- 
suchungen  iiber  die  quantitative  Analyse  des  Traubenzuckers,  ibid.,  vol. 
69,  p.  399;  also  later  volumes  up  to  vol.  129,  p.  362,  1909. 

so  G.  Bertrand,  Le  dosage  des  sucres  reducteurs,  Bull.  Soc.  Chim.,  vol. 
35,  p.  1285,  1906. 

si  Ivar  Bang,  Zur  Methodik  der  Zuckerbestimmung,  Biochcm.  Zeitschr., 
vol.  2,  p.  271,  1906. 


1912]  Birckner:  Glucose  Oxydations  143 

the  glycogen  content  of  animal  organs,82  it  may  be  well  to  call 
attention  to  a  frequent  source  of  error  in  such  determinations 
caused  by  the  iron  content  of  the  respective  organs,  as  was  lately 
pointed  out  by  Starkenstein.83 

Nef  s  latest  view  concerning  the  nature  of  the  products  which 
arise  from  hexoses  under  the  influence  of  Fehling's  solution,  may 
be  found  in  an  article  of  his  pupil  Anderson.84 

Griefenhagen,  Konig  and  Scholl,85  in  an  interesting  investi- 
gation, have  lately  worked  out  a  new  method  of  sugar  deter- 
mination, which  is  based  on  the  observation  that  oxalic  acid  is 
a  regular  oxidation  product  of  all  sugars,  if  they  be  acted  upon 
by  KMnO4  and  alkali,  and  that  its  formation  takes  place  quan- 
titatively. If  'the  sugar  is  a  hexose,  the  process  was  found  to 
follow  the  equation 

C6H12O6  +  5  O2  =  2  H2C2O4  +  2  CO,  +  2  H2O. 

By  using  a  standard  solution  of  KMn04  at  the  start,  the 

amount  of  oxygen  given  off  gives  an  exact  measure  of  the  amount 
of  sugar  present.  The  method  so  far  as  investigated  gave  very 
satisfactory  results. 

The  oxydation  products  of  glucose  in  the  presence  of  the 
halogen  elements  or  of  acids  are  chiefly  those  that  show  no 
rupture  of  the  original  carbon  chain,  viz.,  glucomc  acid  and 
saccharic  acid. 

The  formation  of  glucuronic  acid  under  the  influence  of 
H2028C  has  already  been  mentioned.  Together  with  phenoles  in 
strongly  acid  solutions,  glucose,  as  is  generally  known  to  be  the 
case  with  all  carbohydrates,  gives  rise  to  colored  compounds, 
chiefly  furfurol  derivatives. 


82  See  E.  Pfluger,  Das  Glycogen  (2te.  Aufl.,  Bonn.  1905),  p.  106;  Meine 
Methode  der  quantitativen  Analyse  des  Glycogens,  etc.,  Pfluger 's  Archiv, 
vol.  129,  p.  362,  1909. 

83  G.  Starkenstein,  uber  den  Glyeogenhalt  der  Tunicaten,  nebst  Ver- 
suchen    uber   die   Bedeutung   des   Eisens   fiir   die   quantitative    Glycogen- 
bestimmung,  Biochem.  Zeitschr.,  vol.  27,  p.  53,  1910. 

s*  E.  Anderson,  On  the  Action  of  Fehling's  solution  on  Galatose,  Am. 
Chem.  Journ.,  vol.  42,  esp.  pp.  403-406. 

85  W.  Greifenhagen,  J.  Konig  und  A.  Scholl,  Bestimmung  der  Kohle- 
hydrate  durch  Oxydation  mittels  Kaliumpermanganat  in  alkalischer 
Lb'sung,  Biochem.  Zeitschr.,  vol.  35,  p.  It59,  1911. 

so  See  Jolles,  loc.  cit. 


144        University  of  California  Publications  in  Physiology  [VOL.  4 

H.  Schade87  has  much  endeavored  to  find  chemical  analoga 
of  fermentative  reactions.  According  to  his  studies,  it  is  possible 
by  a  successive  application  of  the  catalytic  influences  of  alkali, 
acid,  and  metal,  to  bring  about  an  alcoholic  fermentation  of 
glucose  by  purely  chemical  means.  He  represents  the  different 
stages  of  the  process  in  the  following  way : 

Dextrose 

(Alkali  as  catalysor) 

Lactic  acid 

(Sulphuric  acid  as  catalysor) 

Acetic  aldehyde  +  formic  acid 
(Ehodium  as  catalysor) 

Alcohol  +  COo 

B.  THE  OXYDATTONS  AND  CLEAVAGES  OF  GLUCOSE  THROUGH  THE 
ACTION  OF  MORE  OR  LESS  UNKNOWN  AGENCIES 

The  phenomena  to  which  reference  will  be  made  in  this  sec- 
tion are  largely  of  a  nature  which  at  the  present  state  of  our 
knowledge  wre  have  no  means  of  explaining  in  a  satisfactory 
way.  There  is  is  a  strong  tendency  to  attribute  most  of  these 
phenomena  to  some  sort  of  fermentative  activity.  But  although 
this  assumption  has  a  certain  degree  of  probability  in  view  of 
the  fact  that  ferment  action  may  be  met  with  outside  of  as  well 
as  inside  of  the  organism,  we  know  of  only  comparatively  few 
cases  in  which  the  presence  of  a  ferment  has  been  demonstrated 
convincingly. 

The  oxydative  destruction  of  glucose  may  either  be  complete 
or  incomplete.  A  complete  cleavage  leads  to  the  formation  of 
CO2,  and  wre  may  include  under  this  heading  the  processes  in- 
volved in  respiration  phenomena  (both  normal  and  intra- 
molecular respiration)  and  the  alcoholic  fermentation.  As  an 
incomplete  oxydation  we  may  regard  the  formation  of  acids. 
This  is,  of  course,  only  an  artificial  classification;  no  sharp  line 
can  really  be  drawn  between  complete  and  incomplete  sugar 
oxydations. 


s?  H.    Schade,   tiber   die   Vorgange    der   Garung   vom    Standpunkt    der 
Katalyse,  BiocJiem.  Zeitschr.,  vol.  7,  p.  299,  1907. 


1912 ]  Birckner:  Glucose  Oxydations  145 

The  process  which  is  known  best,  and  which  has  been  studied 
for  the  longest  time,  is  that  of  alcoholic  fermentation.  Nobody 
will  doubt  any  longer  the  fermentative  character  of  this  process. 
Only  the  nature  of  the  intermediary  stages  of  the  reaction  is  not 
yet  sufficiently  elucidated.  That  the  intramolecular  respiration 
of  living  organisms,  as  far  as  it  concerns  the  sugar,  is  in  all 
chemical  respects  identical  with  the  alcoholic  fermentation  was 
predicted  by  Pasteur88  as  early  as  1872.  It  was  definitely  proven 
by  the  more  recent  investigations  of  Godlewski  and  Polszeniusz,89 
Stoklasa,00  Palladin  and  Kostytschew,91  and  Maximow.92 

As  far  as  the  mechanism  of  the  normal  respiration  is  con- 
cerned, we  may  say  that  we  are  still  in  the  very  beginning 
of  this  study.  That  ferments  are  involved  in  these  processes, 
however  probable,  has  not  at  all  been  sufficiently  proven.  Up 
to  recently  it  was  widely  assumed  that  the  oxygen  is  activated  by 
some  oxygen  catalyst  in  the  respiratory  organs.  At  present, 
however,  it  is  believed  that  the  more  important  prerequisite  of 
sugar  combustion  is  a  far-reaching  dissociation  or  loosening  of 
the  hexose  molecule  itself.  Whichever  may  be  the  real  point  of 
attack,  it  has  become  customary  to  speak  of  this  sugar  trans- 
formation as  of  an  act  of  glucolysis,  and  the  hypothetic  ferment, 
which  is  assumed  to  form  the  active  principle  in  these  changes, 
has  been  termed  the  glucolytic  ferment  of  the  body.  Very  little 
is  known  about  the  individuality  and  the  mode  of  action  of  this 


ss  L.  Pasteur,  Note  au  sujet  d  'une  assertion  de  M.  Fremy,  etc.,  Compt. 
rend.,  vol.  75,  p.  1056,  1872. 

89  Godlewski  und  Polszeniusz,  tiber  intramoleculare  Atmung  (Krakau, 
1901);  cf.  Czapek,  Ergeb.  der  Physiologic,  vol.  9,  p.  606,  1910. 

90  J.  Stoklasa,  Hofm.  Beitrdge  zur  chem.  Physiologic  und  Path.,  vol.  3, 
p.  460,  1902;  Zentralbl.  f.  Physiologic,  vol.  16,  p.  652,  1902;  Ber.  Deutsch. 
chem.  Ges.,  vol.  36,  p.  622,  1903;  ibid.,  p.  4058;  Zentralbl.  f.  Bacteriologie, 
Abt.  II,  vol.  13,  p.  86,  1904;  Ber.  Deutsch.  Bot.  Ges.,  vol.  22,  p.  460,  1904; 
Pfliiger's  Archiv,  vol.  101,  p.  311,  1904;  Zeitschr.  f.  physiol.  Chemie,  vol.  49, 
p.   303,^1907;   Festschrift  fiir  Wiesner,  p.   218,   1908;   Zeitschr.  f.   Zucker- 
industrie  Bohmens,  vol.   32,  p.   273,  1908;    cf.   Czapek,  Ergeb.  d.  Physiol., 
vol.  9,  p.  589,  1910. 

si  W.  Palladin  und  S.  Kostytschew,  Anaerobe  Atmung,  Alokoholgarung 
und  Acetonbildung  bei  den  Samenpflanzen,  Zeitsclir.  f.  Physiol.  Chemie, 
vol.  48,  p.  214,  1906;  Ber.  Deutsch.  Bot.  Ges.,  vol.  24,  p.  273,  1906. 

»2  N.  A.  Maximow,  Zur  Frage  liber  die  Atmung.  Ber.  Deutsch.  Bot    Ges 
vol.  22,  p.  225,  1904. 


146        University  of  California  Publications  in  Physiology  [VOL.  4 

ferment,  and  its  very  existence  has  not  infrequently  been  actually 
questioned.83 

The  incomplete  oxydation  of  glucose  to  acids  is  chiefly  known 
as  a  vital  function  of  many  microbes.  That  these  organisms  act 
by  certain  ferments,  which  they  form  in  their  body,  is  widely 
assumed,  but  only  in  a  very  few  cases,  so  far,  could  a  respective 
glucolytic  ferment  be  isolated.  For  the  lactic  acid  formation  in 
muscle,  the  interaction  of  a  ferment  is  expressively  denied  by 
Fletcher  and  Hopkins94  in  their  recent  communications. 

I  shall  at  first  dwell  upon  a  number  of  cases  in  which  a 
destruction  of  glucose  was  effected  in  organic  fluids  by  some 
agent  or  agents  of  unknown  character,  other  than  cellular 
activity.  Finally,  I  shall  give  a  brief  outline  of  the  sugar 
transformations  that  are  known  to  take  place  in  living  organisms. 

The  discovery  of  Buchner95  that  the  sugar-splitting  prin- 
ciple can  be  extracted  from  the  yeast,  and  that  it  can  transform 
glucose  into  alcohol  and  C02  outside  of  the  cell  is  so  well  known 
in-  its  details  and  in  its  bearing  on  our  present  conceptions  of 
fermentation  problems  that  I  can  refrain,  in  this  review,  of 
giving  a  complete  description  of  this  fundamental  observation.96 

The  next  example,  to  my  knowledge,  of  a  destruction  of  sugar 
in  plant  extracts,  was  furnished  by  M.  Hahn97  in  1900.  Halm 
studied  the  autodigestion  of  a  juice,  which  he  had  obtained  by 
crushing  the  flowering  shafts  of  Arum  maculatum  by  means  of 
a  hydraulic  press  in  the  manner  described  by  Buchner  (loc.  cit.) 
for  yeast.  On  standing  at  room  temperature  the  liquid  showed 
a  rapid  decrease  in  sugar  content,  accompanied  by  a  decrease 
in  weight  and  an  ample  production  of  C02.  The  reaction  was 


93  See  Abderhalden,  Lehrbuch  der  Physiologischen  Chemie  (2  te  Anil. 
Urban  &  Schwarzenberg,  Berlin,  1909),  pp.  107,  596. 

9*  W.  M.  Fletcher  and  F.  G.  Hopkins,  Lactic  acid  in  amphibian  muscle, 
Journ.  Physiol.,  vol.  35,  p.  247,  1906;  W.  M.  Fletcher,  On  the  alleged  for- 
mation of  lactic  acid  in  muscle  during  autolysis  and  in  post-survival 
periods,  Journ.  Physiol.,  vol.  43,  p.  286,  1911. 

95  E.  Buchner,  Alkoholische  Garung  ohne  Hefezellen,  Ber.  Deutsch. 
chem.  Ges.,  vol.  30,  pp.  117,  1110,  1897;  ibid.,  vol.  31,  p.  568,  1898. 

»6  For  method  see  Buchner  und  Hahn,  Die  Zymasegarung,  Miinchen, 
1903.  See  also  the  much  more  simple  method  of  zymase  preparation 
recently  devised  by  von  Lebedew  (Comp.  rendus,  vol.  152,  pp.  49,  1129, 
1911.  Zeitschr.  f.  Physiol.  Chemie,  vol.  73,  p.  447,  1911). 

97  Martin  Hahn,  Chemische  Vorgange  im  Zellfreien  Gewebsaft  von 
Arum  maculatum,  Ber.  Deutsch.  chem.  Ges.,  vol.  33,  p.  3555,  1900. 


1912J  Birckner:  Glucose  Oxydations  147 

greatly  depressed  at  60°  C.  and  was  arrested  after  boiling.  Hahn 
ascribed  these  phenomena  to  the  presence  of  an  oxydative 
ferment. 

In  turning  to  the  animal  kingdom,  we  meet  with  numerous 
researches  for  glucolytic  ferments,  especially  in  the  blood. 
Scheremetjewski98  had  already  observed  that  in  blood,  which 
contained  sugar,  the  oxygen  content  decreased  on  standing,  while 
the  C02  content  increased  correspondingly.  Later  on,  Cl.  Ber- 
nard09 stated  that  from  fresh  blood,  on  standing,  the  sugar  dis- 
appears, and  that  its  place  is  taken  by  lactic  acid.100  Lepine101 
confirmed  this  statement  and  ascribed  the  phenomenon  to  the 
action  of  a  glucolytic  ferment.  Krauss102  also  assumed  such  a 
ferment,  but  at  the  same  time  pointed  out  that  it  could  not  play 
an  important  part  in  sugar  metabolism  as  its  small  efficiency  is 
in  no  proportion  to  the  great  quantities  of  sugar  that  are  con- 
stantly undergoing  change.  Rohmann103  and  Spitzer104  observed 
similar  decompositions  of  sugar  not  only  in  the  blood,  but  in 
aqueous  extractions  of  many  organs.  They  considered  this 
phenomenon,  however,  as  a  function  of  the  living  protoplasm  at 
that  time.  The  experiments  of  N.  Sieber105  showed  the  presence 
of  three  glucolytic  ferments  in  the  fibrine  of  the  blood  plasm. 
The  three  fractions  could  be  obtained  as  powders,  and  showed 
some  of  the  typical  reactions  of  the  oxydases.  This  author, 
therefore,  is  inclined  to  consider  the  oxydases  as  of  great  bio- 


»8  Scheremetjewski,  tiber  die  Anderung  des  respiratorischen  Gasaus- 
tausches  durch  d.  Zufiigung  verbrennlicher  Molecule  zum  kreisenden 
Blute,  Sachs.  Ges.  Wissensch.  Math.  phys.  Kl.  (Leipzig,  1868),  vol.  20,  p. 
154. 

99  Claude  Bernard,  Legons  sur  le  diabete  (Paris,  1877),  p.  128. 

100  On  the  latter  statement   cf.   T.   Launder   Brunton,   On   a   probable 
glycolytic  ferment  in  muscle,  etc.,  Zeitschr.   f.  Biologie,  vol.   34,  p.  487, 
1896. 

101  R.  Lepine,  several  articles  in  Comptes  rendus  de  I'Acad.  des  Sciences, 
1890-1895;    cf.    Abderhalden,   Lehrbuch   der  Physiologischen   Chemie,   2  te. 
Aufl.,  1909,  p.  89. 

102  F.  Krauss,  Zeitschr.  f.  klinische  Medisin,  vol.  21,  p.   315,  1892;   cf. 
N.  Sieber,  Zeitschr.  f.  Physiol.  Chemie,  vol.  39,  p.  507,  1903. 

103  Zentralblatt.    f.    mediz.    Wissenschaften,    vol.    51,    p.    849,    1893;    cf. 
Sieber,  loc.  cit. 

104  Berliner  klinische  Wochenschrift,  1894,  p.  949;  cf.  Sieber,  loc.  cit. 

105  N.    Sieber,   Einwirkung   der   Oxydationsenzyme    auf   Kohlehydrate, 
Zeitschr.    f.   Physiol.   Chemie,   Vol.    39,    484,    1903;    Zur   Frage    nach    dem 
glyeolytischen  Princip  des  Blutfibrins,  idem.,  vol.  44,  p.  560,  1905. 


148        University  of  California  Publications  in  Physiology  [VOL.  4 

logical  importance.  The  progress  of  the  reaction  was  measured, 
3is  in  previous  investigations,  by  the  disappearing  of  the  sugar 
and  the  simultaneous  formation  of  C02  Of  the  three  fractions, 
the  alcohol  soluble  part  especially  showed  a  marked  resistence 
against  heat. 

For  muscular  tissues,  Claude  Bernard106  showed  that  not  only 
do  dead  muscles  become  acid  at  the  expense  of  the  sugar  and 
glycogen  they  contain,  but  that  they  also  cause  the  formation  of 
acid  in  a  solution  of  grape-sugar  to  which  they  are  added. 
Brunton  (loc.  cit.)  is  the  first,  in  a  brief  note,  to  mention  a 
glucolytic  ferment  of  the  muscle.  Cohnheim107  succeeded  in 
extracting  the  inactive  form  of  this  ferment,  and  showed  that  it 
could  be  activated  by  a  co-ferment  which  he  obtained  from  an 
alcoholic  extraction  of  the  pancreas.108  Cohnheim  chiefly  studied 
the  relation  between  the  amount  of  co-ferment  added,  and  the 
extent  of  the  sugar,  destruction.  He  noticed  the  formation  of 
small  quantities  of  acids,  including  slight  amounts  of  C02  gas, 
leaving,  however,  the  question  of  the  products  open  for  its  main 
part. 

Levene  and  Meyer,100  in  a  recent  attempt  to  clear  up  this 
point,  failed  to  detect  carbonic,  formic,  acetic  or  lactic  acids 
among  the  products  resulting  from  this  apparent  disappearance 
of  glucose.  In  their  belief,  the  latter  phenomenon  is  due  to  a 
condensation  process  (formation  of  a  di-saccharide)  under  the 
influence  of  the  combined  tissue  extracts. 

Sieber  and  Dzierzgowski110  claim  that  the  cell-free  liquid  that 


100  Cf .  Brunton,  loc.  cit. 

107  Cohnheim,  Die  Kohlehydrateverbremmng  in  den  Muskeln,  und  ihre 
Beeinflussung  durch  das  Pancreas,  Zeitschr.  f.  Physiol.  Chemie,  vol.*39, 
p.  336,  1903;  ibid.,  vol.  42,  p.  401,  1904;  tiber  Glycolyse,  ibid.,  vol.  47, 
p.  253,  1906. 

!os  Stoklasa's  recent  investigations  (J.  Stoklasa,  tiber  die  glucoly- 
tischen  Enzyme  im  Pancreas,  Zeitschr.  f.  Physiol.  Chemie,  vol.  62,  p.  36, 
1909)  have  definitely  shown  that  from  the  pancreas  itself  (pig's  pancreas) 
no  ferment  can  be  extracted,  that  would  have  some  action  on  hexoses. 
See  also  G.  W.  Hall,  Concerning  Glycolysis,  Am.  Journ.  Physiol.,  vol.  18, 
p.  283,  1907. 

io»  P.  A.  Levene  and  G.  M.  Meyer,  Journ.  Biol.  Chem.,  vol.  9,  p.  97, 
1911;  ibid.,  vol.  11,  p.  347,  1912. 

no  N.  Sieber  und  W.  Dzierzgowski,  Die  Enzyme  der  Lunge,  Zeitschr. 
f.  Physiol.  Chemie,  vol.  62,  p.  263,  1909. 


1912]  Birckner:  Glucose  Oxydations  149 

can  be  obtained  by  crushing  the  lungs  of  horses,  even  after 
previously  washing  out  all  the  blood,  contains,  among  others, 
a  glucolytic  ferment,  while  Levene  and  Meyer111  report  a  different 
result  with  the  tissues  of  other  animals.  Finally,  I  may  mention 
an  observation  made  by  Vandevelde.112  If  to  normal  urine 
glucose  was  added,  and  the  mixture  kept  at  37°  C.  under  sterile 
conditions,  a  distinct  fall  of  the  optical  activity  was  observed 
after  one  year's  standing,  while  the  reducing  power  against 
Fehling's  solution  remained  unaltered.  The  same  observation 
wras  made  with  diabetic  urine  (no  addition  of  sugar).  The 
phenomenon,  I  may  infer,  is  perhaps  due  to  the  formation  of  an 
inactive  combination  product  between  glucose  and  urea.113 

All  these  investigations  which  are  confined  to  the  measure- 
ment of  the  disappearance  of  sugar  or  of  the  consumption  of 
oxygen  in  connection  with  a  production  of  C02,  do,  however,  not 
approach  the  real  problem  itself.  We  know  sufficiently  well 
that  sugar  is  constantly  being  broken  down  in  the  organism, 
and  that  C02  and  water,  or  C02  and  alcohol,  respectively,  are 
the  end-products  of  these  transformations.  The  real  problem,  is, 
however,  to  find  out  by  what  means  the  living  substance  brings 
about  these  rapid  transformations,  or,  as  the  first  step  towards 
this  aim,  to  find  the  intermediary  stages  in  these  oxydations. 
C02,  and  even  alcohol  may  be  readily  formed,  according  to  recent 
investigations,  from  dead  organic  materials,114  even  from  nitrogen 
containing  substances,115  either  spontaneously  or  through  the 
action  of  yeast  (the  latter  case  referring  to  the  alcoholic  fer- 


111  P.  A.  Levene  and  G.  M.  Meyer,  Journ.  Biol.  Chem.,  vol.  11,  p.  353, 
1912. 

112  A.  J.  J.  Vandevelde,   Polarimetrisch  messbare  Zuckerzerstorungen 
in  physiologischen  Fliissigheiten,  Biochem.  Zeitschr.,  vol.  23,  p.  324,  1909. 

us  See  M.  N.  Schoorl,  Les  ureides  (earbamides)  des  sucres,  Eec.  des 
trav.  des  Pays-Bas  et  de  la  Belg.,  vol.  22,  p.  1,  1903;  also  P.  Mayer,  tiber 
Ureidoglucose,  Biochem.  Zeitschr.,  vol.  17,  p.  145,  1909. 

114  See  F.  Czapek,  Ergeb.  d.  Physiol.,  vol.  9,  p.  600,  1910,  and  references 
on  that  page. 

us  See  F.  Ehrlich,  tiber  eine  Methode  zur  Spaltung  racemischer  Amino- 
sauren  mittels  Hefe,  Biochem.  Zeitschr.,  vol.  1,  p.  8,  1906;  ibid.,  vol.  8, 
p.  438,  1908;  also  O.  E.  Ashdown  and  J.  T.  Hewitt,  The  by-produets  of 
alcoholic  fermentation,  Journ.  Chem.  Soc.,  vol.  97,  p.  1636,  1910;  O.  Neu- 
bauer  and  K.  Fromherz,  tiber  den  Abbau  der  Aminosauren  bei  der  Hefe- 
garung,  Zeitschr.,  f.  Physiol.  Chemei,  vol.  70,  p.  326,  1910;  C.  Neuberg  and 
others,  tiber  Zuckerfreie  Hefegarungen,  Biochem.  Zeitschr.,  vol.  31,  p. 
170,  1911;  ibid.,  vol.  32,  p.  323,  1911;  ibid.,  vol.  36,  p.  60,  1911. 


150        University  of  California  Publications  in  Physiology  [VOL.  4 

mentation  of  nitrogen  compounds),  so  that  the  observations  to 
which  I  have  referred  above  do  not  give  us  any  essentially  new 
information. 

As  to  the  intermediary  stages  of  the  alcoholic  fermentation, 
our  conceptions  have  undergone  various  changes  in  recent  years. 
Very  likely  we  shall  have  to  encounter  two  or  more  intermediate 
products  in  this  process,  resulting  from  two  or  more  coordinate 
reactions  which  proceed  independently  of  one  another  and  each 
of  which  is  catalyzed  by  a  different  constituent  of  the  zymase 
preparation. 

Although  the  assumption  of  an  intermediate  formation  of 
lactic  acid110  is  now  generally  abandoned,117  the  formation  of  a 
substance  closely  related  to  it  is  very  probable.118  Certain  yeasts 
have  the  faculty  of  producing,  during  the  normal  fermentation 
process,  marked  amounts  of  formic  acid,  and  this  process  is 
regarded  as  an  intermediary  step  of  the  alcoholic  fermentation 
by  some  authors.119 

Furthermore,  the  intermediate  formation  of  ester-like  com- 
binations between  glucose  (and  perhaps  also  between  a  resulting 
triose)  and  phosphate  groups  seems  to  be  a  very  important  factor 
in  the  process  of  alcoholic  fermentation,  and  phosphoric  acid  in 


us  E.  Buchner  und  J.  Meisenheimer,  Die  chemischen  Vorgange  bei  der 
alkoholischen  Garung,  Ber.  Deutsch.  diem.  Ges.,  vol.  37,  p.  419,  1904;  ibid., 
vol.  38,  p.  620,  1905.  A.  Wohl,  Die  neueren  Ansichten  iiber  den  chemischen 
Verlauf  der  Garung,  Blocliem.  Zeitschr.,  vol.  5,  p.  45,  1907. 

117  E.  Buchner  und  J.  Meisenheimer,  Die  chemischen  Vorgange  bei  der 
alcoholischen  Garung,  iv,  Ber.  Deutsch.  chem.  Ges.,  vol.  43,  p.  1773,  1910. 

us  P.  Boysen  Jensen,  Die  Zersetzung  des  Zuckers  wiihrend  des  Kes- 
pirationsprocesses,  Ber.  Deutsch.  Bot.  Ges.,  vol.  26a,  p.  666,  1908.  Sukker- 
s0nderdelingen  under  Eespirationsprocessen  hos  H0jere  Planter,  Dissert. 
Copenhagen,  1910. 

us  See  for  instance:  H.  Franzen,  Tiber  die  Vergarung  und  Bildung  der 
Ameisensaure  durch  Hefen,  Zeitschr.  f.  Physiol.  Chemie,  vol.  77,  p.  129, 
1912. 


1912J  Birckner:  Glucose  Oxydations  151 

organic  combination  (e.g.,  as  lecithin)  is  looked  upon  as  the 
activating  complement  of  zymase.130 

Recently,  Harden  and  Young,121  von  Lebedew,122  and  still 
later  Euler  and  his  coworkers,123  have  studied  more  closely  the 
structure  of  these  hexose  phosphates,  and  the  manner  in  which 
they  are  hydrolyzed  again. 

Harden  and  Young  represent  this  phosphate  cycle  of  the 
alcoholic  fermentation  in  the  following  way : 

(I)     2  C,HM0.  +  2  R'2HP04    >  2  C02  +  2  C2H6O  +  C6H10O4(PO4E',)2 

+  H20. 

(II)      C8H1004(P04R'2)2  +  2  tt,0 >    C6H1206  +  2  R'2HPO4. 

Reaction  (II),  according  to  these  authors,  is  brought  about 
by  a  special  enzyme  which  they  call  "hexose  phosphatase. " 
This  whole  formulation  is  strongly  opposed,  however,  by  von 
Lebedew  (loc.  cit.}. 

According  to  Kostytschew  and  Scheloumow,124  the  stimulating 
effect  of  the  phosphate  salt  is  perhaps  due  only  to  its  alkaline 
reaction. 


120  Numerous  articles  by: 

A.  Harden  and  W.  J.  Young,  Proc.  Chem.  Soc.,  vol.  21,  p.  189,  1905; 
ibid.,  vol.  24,  p.  115,  1908;  Soy.  Soc.  Proc.  (B),  vol.  77,  p.  405,  1906; 
ibid.,  vol.  80,  p.  299,  1908;  ibid.,  vol.  81,  p.  336,  1909;  ibid.,  vol.  82,  p. 
321,  1910;  Centralbl.  f.  Bacterial.,  (n)  vol.  26,  p.  178,  1910;  Biochem. 
Zeitschr.,  vol.  32,  p.  173,  1911. 

W.  J.  Young.  Proc.  Chem.  Soc.,  vol.  23,  p.  65,  1907;  Roy.  Soc.  Proc. 
(B),  vol.  81,  p.  528,  1909;  Biochem.  Zeitschr.,  vol.  32,  p.  177,  1911. 

H.  Euler  und  G.  Lundeqvist,  Zeitschr.  f.  Physiol.  Chemie,  vol.  72, 
p.  97,  1911. 

L.  Iwanoff,  Trav.  Soc.  des  Natur.  de  St.  Petersbourg,  vol.  34,  1905, 
cf.  Euler  und  Ohlsen,  below;  Zeitschr.  f.  Physiol.  Chemie,  vol.  50,  p.  281, 
1906;  Centralbl.  f.  Bacterial,  (n),  vol.  24,  p.  1,  1909;  Biochem.  Zeitschr., 
vol.  25,  p.  171,  1910. 

E.  Buchner  und  J.  Meisenheimer,  loc.  cit. 

E.  Buchner  und  W.  Albertoni,  Zeitschr.  f.  Physiol.  Chemie,  vol.  46, 
p.  136,  1905. 

121  Biochem.  Zeitschr.,  vol.  32,  p.  173;  and  references  quoted  sub  W.  J. 
Young. 

122  A.  von  Lebedew,  Versuche  zur  Aufklarung  des  zellfreien  Garungs- 
processes  mit  Hilfe   des  Ultrafilters,   Biochem.  Zeitschr.,  vol.   20,   p.   114, 
1909;  Tiber  Hexosephosphorsaureester,  Biochem.  Zeitschr.,  vol.  28,  p.  213, 
19lO;ibid.,  vol.  36,  p.  248,  1911. 

12s  H.  Euler  und  A.  Fodor,  fiber  ein  Zwischenprodukt  der  alcoholischen 
Garung,  Biochem.  Zeitschr.,  vol.  36,  p.  401,  1911;  H.  Euler  and  H.  Ohlsen, 
Tiber  den  Einfluss  der  Temperatur  auf  die  Wirkung  der  Phosphatese, 
ibid.,  vol.  37,  p.  133,  1911. 

124  S.  Kostytschew  und  A.  Scheloumow,  Tiber  die  Einwirkung  der 
Garungsprodukte  und  der  Phosphate  auf  die  Pflanzentamung,  Jahrb.  f. 
wiss.  Botanik,  vol.  50,  p.  157,  1911. 


152        University  of  California  Publications  in  Physiology  [VOL.  4 

Boysen  Jensen  (loc.  cit.)  obtained  evidence  for  the  presence 
of  two  forms  of  dioxyacetone,  an  isomeride  of  lactic  acid,  in  the 
glucose-zymase  digest  under  certain  experimental  conditions. 
He  regards  dioxyacetone  as  a  regular  intermediate  product  of 
both  the  alcoholic  fermentation  and  the  normal  respiration,  in 
accordance  with  the  following  scheme : 


(Dextrase)                                          (Oxydase) 
Dextrose >  Dioxyacetone >  CO2  +  H2O 


C02  +  C2H5OH 

The  ferment  zymase,  it  is  seen,  consists  of  the  two  fractions 
dextrase  and  dioxyacetonase.  Besides,  after  the  arrival  at  the 
dioxyacetone  stage,  an  oxdyase  may  come  into  action  (oxydases 
are  not  known  to  attack  either  glucose  or  alcohol). 

We  may  therefore  imagine  that,  depending  on  whether  the 
respective  organism  contains  primarily  the  ferment  oxydase  or 
the  ferment  dioxyacetonase,  the  second  half  of  the  reaction  will 
either  be  an  act  of  normal  respiration,  or  an  alcoholic  fermen- 
tation. Higher  organisms  have  largely  lost  the  power  of  form- 
ing dioxyacetonase,  and  therefore  their  subsistence  depends  on 
the  presence  of  oxygen,  while  in  some  lower  organisms,  with  high 
content  of  dioxyacetonase  as  compared  with  the  oxydase,  the 
alcoholic  fermentation  is  predominant.  Many  of  the  lower 
fungi  (e.g.,  yeasts)  have  the  faculty  of  forming  both  ferments 
simultaneously,  and  in  their  life,  the  normal  respiration  and 
the  alcoholic  fermentation  proceed  side  by  side.  Others  (e.g., 
Mucor,  and  even  phaenogamic  plants  and  lower  animals),  which 
normally  are  consumers  of  atmospheric  oxygen,  may  become 
producers  of  alcohol  in  case  of  any  shortage  in  the  oxygen 
supply  (phenomena  of  intramolecular  respiration).  Boysen 
Jensen  has  been  able  to  prove  the  formation  of  dioxyacetone 
directly.  It  is  also  known  that  dioxyacetone  is  readily  ferment- 


1912]  Birckner:  Glucose  Oxydations  153 

able,125  while  the  other  three  substances  which  were  temporarily 
regarded  as  intermediary  products  (lactic  acid,  methyl  gly- 
oxaline,  and  glycerose),  are  not,  or  only  difficultly  (glycerose), 
fermented  by  yeasts.126 

W.  Lob12T  finds  Jensen's  results  in  agreement  with  his  own 
theory. 

Apart  from  these  cases,  the  instances  in  which  products  of 
fermentative  sugar  oxydations  have  been  obtained,  other  than 
CO2,  H20,  and  alcohol,  are  very  few  in  number.  Buchner  and 
Meisenheimer128  have  ascertained  the  presence  of  a  ferment  in 
Bac.  Delbriicki  (syn.  Bac.  acidificans  longissimus}  which  is  able 
to  split  glucose  into  two  molecules  of  dl-lactic  acid. 

Weinland129  demonstrated  the  presence  of  an  enzyme  in  the 
clear  "press- juice"  of  Ascaris  lumbicoides  which  splits  glucose 
into  valerianic  acid,  carbon  dioxyde,  and  hydrogen. 

W.  Lob130  succeeded  in  showing  that  the  alcohol-soluble  part 
of  pig's  pancreas,  if  brought  into  a  firm  combination  with  iron, 
will  act  on  glucose  in  a  way  similar  to  the  action  of  peroxydases. 
As  among  the  products  of  this  glucolysis,  he  obtained  small 
amounts  of  C02,  formic  acid,  formaldehyde  (traces),  and  pentose. 

I  have  myself  succeeded  in  obtaining  a  ferment  preparation 
from  yeast,  which  shows  considerable  glucolytic  activity,  prefer- 
ably at  an  elevated  temperature.  It  causes  no  gas  formation 
and  furnishes  a  solid,  carbon-like  substance  as  a  result  of  pro- 
longed action.  Among  the  products  are  acids,  but  I  have  also 
been  able  to  ascertain  the  formation  of  formaldehyde  and  pen- 


125  G.  Bertrand,  Etude  biochimique  de  la  bacterie  du  sorbose,  Ann.  de 
chim.  et  de  phys.,  (8),  vol.  3,  p.  187,  1904;  Buchner  and  Meisenheimer,  Ber. 
Deutsch.  chem.  Ges.,  vol.  43,  p.  1773,  1910.  See,  however,  also  A.  Slator, 
Ber.  Deutsch.  chem.  Ges.,  vol.  45,  p.  43,  1912. 

120  Cf.  from  E.  L.  Pinner,  Fortschritte  der  Garungschemie,  Fortschr. 
der  Chemie,  Physik  und  Physical.  Chemie,  vol.  4,  p.  135,  1911. 

IK  Biochem.  Zeitschr.,  vol.  29,  p.  311,  1910. 

128  E.  Buchner  und  J.  Meisenheimer,  Enzyme  bei  Spaltpilzgarungen, 
Ber.  Deutsch.  chem.  Ges.,  vol.  36,  p.  634,  1903;  tiber  die  Milchsauregarung, 
Liebig's  Ann.  d.  Chemie,  vol.  349,  p.  125,  1906. 

120  E.  Weinland,  tiber  Kohlehydratzersetzung  ohne  Sauerstoffaufnahme 
bei  Ascaris,  einen  tierischen  Garungsprocess,  Zeitschr.  f.  Biologic,  vol.  42, 
p.  55,  1901;  tiber  ausgepresste  Extrakte  von  Ascaris  lumbicoides  und  ihre 
Wirkung,  ibid.,  vol.  43,  p.  86,  1902;  tiber  die  von  Asc.  lumbicoides  aus- 
geschiedene  Fettsaure,  ibid.,  vol.  45,  p.  113,  1903. 

iso  \v.  Lob  und  Pulvermacher,  Biochem.  Zeitschr.,  vol.  29,  p.  316,  1910. 


154        University  of  California  Publications  in  Physiology  [VOL.  4 

tose.  A  report  of  the  work  is  given  in  the  second  part  of  this 
paper. 

While  all  the  authors  so  far  quoted  in  this  section  considered 
these  glucolytic  ferments  as  being  of  the  general  type  of  oxydases, 
Euler131  objects  to  this  classification.  According  to  him  the 
oxydase  reactions,  which  these  preparations  mostly  give  at  the 
same  time,  have  no  relations  to  their  glucolytic  qualities.  As 
the  latter  function  is  of  primary  importance,  and  as  in  this 
respect,  all  these  ferments  resemble  Buchner's  zymase,  Euler 
unites  all  glucolytic  ferments  under  the  heading  "Garungs- 
enzyme,"  separating  them  from  both  the  oxydases  and  the 
hydrolytic  ferments. 

Little  more  needs  to  be  said  about  the  cleavages  of  glucose 
in  living  organisms.  Glucose  in  the  animal  body  is  the  main 
source  of  muscular  and  of  respiratory  energy.  The  places  in 
which  its  oxydation  transformation  is  going  on,  for  its  main 
part,  are  the  blood,  the  lungs,  and  the  muscles.  Apart  from 
the  fact  that  the  final  products  of  these  transformations  are 
C02  and  H20,  their  chemical  nature  is  very  little  understood. 
In  vitro,  we  are  familiar  with  three  principal  types  of  sugar 
disintegrations,  namely, 

(1)  Direct  Oxydations  (without  cleavages  of  the  mole- 
cule ;  resulting  in  the  formation  of  gluconic  and  saccharic 
acids)  ; 

(2)  Depolymerizations   (splitting  off  of  one  or  more 
formaldehyde  groups.    Reversion  of  photosynthesis)  ; 

(3)  Cleavages    (resulting  in  the  formation  of  lactic 
acid  or  its  homologes. 

Theoretically,  each  of  these  three  types  might  be  involved  in 
the  sugar  combustion  in  living  tissues,  as  by  means  of  secondary 
processes  C02  and  H20  (or  alcohol)  could  easily  be  the  final 
products  of  each  of  these  reactions.  As  far  as  we  know,  true 
cleavage  processes  resulting  in  the  intermediary  formation  of 
lactic  acid  are  of  predominating  importance  in  the  sugar  meta- 
bolism of  animals.  Levene  and  Meyer132  have  just  found  that 


131  Allgemeine  Chemie  der  Enzyme,  p.  37. 

132  p.  A.  Levene  and  G.  M.  Meyer,  Journ.  Biol.  Chem.,  vol.  11,  p.  361, 
1912. 


1912J  Birckner:  Glucose  Oxydations  155 

the  leucocytes  of  the  blood  play  a  very  prominent  part  in  these 
transformations. 

Grlucuronic  acid,  which  in  combinations  forms  a  constituent 
of  many  organs  and  tissue  fluids,  is  perhaps  derived  from 
glucose  by  an  oxydative  process  the  nature  of  which  is  presently 
unknown. 

We  possess  a  far  more  extensive  knowledge,  however,  of  the 
metabolic  sugar  cleavages  in  lower  plants,  and  in  bacteria. 
Here  again  we  would  have  to  mention  yeasts  in  the  first  place, 
the  study  of  which  is  a  constant  source  of  valuable  information.133 

The  genetic  relationship  between  the  alcoholic  fermentation 
(anaerob  respiration)  and  the  respiration  of  higher  organisms 
with  regard  to  glucose  was  early  recognized  by  Pfeffer,134  and 
has  been  clearly  established  through  the  newer  researches 
of  Godlewski,135  Palladin,130  Kostytschew,137  Walther  Lob,138 
Zaleski,13"  and  Boysen  Jensen.140  For  an  excellent  review  of  the 


133  For  the  kinetics  of  alcoholic  fermentation,  see  M.  J.  H.  Aberson, 
La  fermentation  alcoolique,  Eec.  d.  trav.  chim.  des  Pays-Bas,  vol.  22,  p.  78, 
1903,  and  H.  Euler,  Chemische  Dynamik  der  Zellfreien  Garung,  Zeitschr. 
f.  Physiol.  Chemie,  vol.  44,  p.  53,  1905. 

134  \v.    Pfeffer,    Das    Wesen   und    die    Bedeutung    der   Atmung,    Land- 
wirtschaftl.  Jahrbiicher,  vol.  7,  p.  805,  1878. 

135  E.  Godlewski,  Bull,  intern,  de  I'Acad.  des  Sc.  de  Cracovie,  1904,  p. 
115;  cf.  Euler,  Pflanzenchemie,  vol.  2,  p.  172,  1909.     See  below. 

i3u  \y.  Palladin,  tiber  den  verschiedenen  Ursprung  der  wahrend  der 
Atmung  d.  Pflanzen  ausgeschiedenen  Kohlensaure,  Ber.  Deutsch.  Bot.  Ges., 
vol.  23,  p.  240,  1905;  Bildung  d.  verschiedenen  Atmungsenzyme  in  Abhan- 
gigkeit  von  dem  Entwicklungsstadium  der  Pflanzen,  ibid.,  vol.  24,  p.  97, 
1906;  tiber  das  Wesen  der  Pflanzenatmung,  Biochem.  Zeitschr.,  vol.  18, 
p.  151,  1909. 

i3T  S.  Kostytschew,  tiber  die  Alkoholgarung  von  Aspergillus  niger,  Ber. 
Deutsch.  Bot.  Ges.,  vol.  25,  p.  44,  1907;  Zur  Frage  der  Wasserstoffbildung 
bei  der  Atmung  d.  Pflanzen,  ibid.,  p.  178;  uber  anaerobe  Atmung  ohne 
Alkoholbildung,  ibid,,  p.  188;  Zweite  Mitt,  uber  anaerobe  Atmung  ohne 
Alkoholbildgun,  ibid.,  vol.  26a,  p.  167,  1908;  tiber  den  Zusammenhang  der 
Sauerstoffatmung  der  Samenpflanzen  mit  der  alkoholischen  Garung,  ibid., 
p.  565;  tiber  die  Anteilnahme  der  Zymase  am  Atmungsprozesse  der  Samen- 
pflanzen, Biochem.  Zeitschr.,  vol.  15,  p.  164,  1908;  tiber  den  Vorgang  der 
Zuckeroxydation  bei  der  Pflanzenatmung,  Zeitschr.  f.  Physiol.  Chemie, 
vol.  67,  p.  116,  1910;  besides,  together  with  W.  Palladin,  Zeitschr.  f. 
Physiol.  Chemie,  vol.  48,  p.  214,  1906. 

138  LOC.    dt. 

139  W.  Zaleski,  Zum  Studium   der  Atmungsenzyme  der  Pflanzen,  Bio- 
chem. Zeitschr.,  vol.  31,  p.  195,  1911;  W.  Zaleski  und  A.  Reinhard,  Unter- 
suchungen  iiber  die  Atmung  der  Pflanzen,  Biochem.  Zeitschr.,  vol.  35    p. 
228,  1911. 

1*0  Loc.  cit. 


156        University  of  California  Publications  in  Physiology  [VOL.  4 

recent  advances  along  these  lines  we  are  indebted  to  Euler.141 

It  remains  to  enumerate  briefly  the  action  on  glucose  of  some 
of  the  more  important  micro-organisms. 

One  of  the  typical  oxydizing  bacteria  is  the  Bacterium 
xylinum  (Adrian  Brown),  which  is  more  commonly  known  from 
the  studies  of  Bertrand142  as  the  "sorbose  bacterium."  Besides 
other  activities,  this  microbe  is  able  to  oxydize  glucose  to 
gluconic  acid.  This  fermentation  was  first  observed  by  Bou- 
troux,143  who  called  the  microbe  Micrococcus  oblongus. 

All  other  vital  fermentations,  so  far  as  known,  result  in  a 
cleavage  of  the  glucose  molecule.  We  have  already  referred  to 
the  important  group  of  bacteria  that  split  glucose  into  two 
molecules  of  lactic  acid.  These  microbes  are  of  general  occur- 
rence in  most  organic  food  materials,  especially  in  milk  and 
certain  beverages  derived  from  it,  such  as  the  well-known 
"kefir"  or  the  Bulgarian  "yoghurt."144  They  have  also  been 
found  to  be  active  in  the  stomach  of  higher  animals  in  cases  of 
carcinoma.145 

That  the  living  leucocytes  of  the  blood  have  the  power  of 
breaking  down  glucose  into  d-lactic  acid  is  one  of  the  latest 
results  obtained  by  Levene  and  Meyer.146  Besides,  there  have 
been  observed  organisms  which  effect  the  formation  from  glucose 
of  the  following  substances. 


141  H.  Euler,  Grundlagen  und  Ergebnisse  der  Pflanzenchemie  (Braunsch- 
weig, Fr.  Vieweg  u.  Sohn,  1909)  2nd  and  3d  part,  p,  171  and  following. 

142  G.  Bertrand,  Action  de  la  bacterie  du  sorbose,  etc.,  Compt.  rend.r 
vol.  126,  p.  984,  1898;  ibid.,  vol.  127,  p.  124,  1898;  La  bacterie  du  Sorbose, 
Ann.  Chim.  Phys.  (8),  vol.  3,  p.  181,  1904. 

1*3  L.  Boutroux,  Sur  une  fermentation  nouvelle  du  glucose,  Compt.  rend 
vol.  91,  p.  236,  1880. 

!44  See,  for  instance,  G.  Bertrand  und  F.  Duchacek,  tiber  die  Einwirkung 
des  Bacillus  bulqaricus  auf  verschiedene  Zuckerarten,  Biochem.  Zeitsclir., 
vol.  20,  p.  100,  1909. 

145  Sandberg,  Zeitschr.  f.  klin.  Medisin.,  vol.  51,  p.  80,  1903;  cf.  Emmer- 
ling,  Biochem.  Centralbl.,  vol.  9,  p.  408,  1909. 

i4c  p.  A.  Levene  and  G.  M.  Meyer,  Journ.  Biol.  Chem.,  vol.  11,  p.  361> 
1912. 


1912] 


Birckner:  Glucose  Oxydations 


157 


Fermentation  products 
Oxalic  acid 

Citric  acid 


Acetic  acid 
Succinie  acid 

Propionic  acid 
Butyric  acid 

Propyl-alcohol 
n-butyl-alcohol 

Butylene-glycol 
Acetyl-methylcarbinol 


See  for  reference : 

W.  Zopf ,  Ber.  Deutsch.  Bot.  Ges.,  vol.  18,  p.  32,  1900. 
G.  Wehmer,  Bot.  Zeitung,  1891,  p.  233. 
G.  Wehmer,  ChemiJcer  Ztg.,  1897,  p.  1022. 

P.  Maze  and  A.  Perrier,  Ann.  de  I'Inst.  Pasteur, 
vol.  18,  p.  553,  1904. 

E.   Buchner  and  Wiistenfeld,  Biochem.  Zeitschr., 
vol.  17,  p.  395,  1909. 

Gayon  and  Dubourg,  Ann.  de.  I'Inst.  Pasteur,  vol. 
15,  p.  527,  1901. 


O.  Emmerling,  Ber.  Deutsch.  chem.  Ges.,  vol.  37, 
p.  3535,  1904. 

Harden  and  Walpole,  Proc.  Boy.  Soc.  London  (B), 
vol.  77,  pp.  399,  519,  1907;  vol.  83,  p.  272,  1911. 


For  more  complete  references  to  this  paragraph  see : 

O.  Emmerling,  Die  Zersetzung  stickstofffreier  organischer  Sub- 
stanzen  durch  Bakterien  (Braunschweig,  Fr.  Vieweg,  1902);  Neuere 
Arbeiten  auf  dem  Gebiet  der  Bakteriengarungen,  Biochemisches 
Zentralblatt,  vol.  9,  p.  397,  1909. 

E.  von  Lippmann,  Die  Chemie  der  Zuckerarten  (Ed.  3,  Braun- 
schweig, Fr.  Vieweg  &  Sohn,  1904),  p.  374  and  following. 


158        University  of  California  Publications  in  Physiology  [Voi,.  4 


PART  II 

YEAST  GLUCASE,  A  NEW  GLUCOLYTIC  FERMENT 

On  the  preceding  pages  I  have  at  several  points  alluded  to 
a  fermentation  phenomenon  which  I  first  happened  to  observe 
some  time  ago,  and  to  the  study  of  which  I  have  lately  devoted 
considerable  time.  A  preliminary  report  on  this  work  may 
follow : 

THE  FERMENT  AS  FIRST  OBSERVED  AND  RECOGNIZED 

In  the  early  part  of  1911,  following  a  suggestion  of  Dr.  T. 
Brailsford  Robertson,  I  found  it  necessary  for  a  certain  purpose 
to  prepare  the  ferment  maltase.  I  tried  to  obtain  it  from 
yeast  by  the  well-known  method  of  Croft  Hill1  or  with  a  slight 
modification  according  to  0.  Emmerling.2  The  material  chosen 
was  the  yeast  of  the  so-called  California  "steam  beer,"  a  local 
brew,  which  although  a  bottom  fermentation  beer,  differs  in 
many  respects  from  the  common  lager  beers. 

Steam  beer  originated  (in  San  Jose)  soon  after  the  discovery 
of  gold  in  California,  and  as  its  characteristic  quality  is  rapidity 
of  preparation,  we  may  infer  that  it  was  intended  chiefly  for  the 
purpose  of  meeting  the  strong  demands  of  those  "early  days." 
Up  to  about  twenty  years  ago,  steam  beer  was  practically  the 
only  beer  produced  on  this  coast. 

The  differences  between  lager  beer  and  steam  beer  depend 
in  the  first  place  on  the  difference  in  temperature  at  which  the 
fermentation  of  the  wort  is  carried  on,  and  they  are  therefore 


1  A.  Croft  Hill,  Reversible  zymo-hydrolysis,  Journ.  Cliem.  Soc.,  vol.  73, 
p.  634,  1898.    For  the  method  see  also  Euler,  Allegmenie  Cliemie  der  Enzyme 
(Wiesbaden,  1910),  p.  15. 

2  0.  Emmerling,  Synthetische  Wirkung  der  Hefemaltase,  Ber.  Deutsch. 
chem.  Ges.  vol.  34,  p.  602. 


Birckner:  Yeast  Glucase  159 

a  direct  function  of    the  respective  metabolic  activity  of    the 
yeast.    The  following  table  will  serve  to  illustrate  these  relations : 


Temp, 
at 
start 

Temp,  maximum 
which  is 
allowed 

Time  required 
for  the  whole 
ferm.  process 

5°C. 

10-11°  C. 

8  to  10  days 

13°  C. 

18°  C. 

3  days 

Lager  beer 
California  ' '  Steam ; 


Besides  the  temperature,  it  is  probably  the  more  extensive 
aeriation  of  the  steam-beer  yeast  which  in  part  causes  its  high 
activity.  After  having  reached  the  temperature  of  18°  C.,  instead 
of  being  cooled  down  by  artificial  means  in  the  fermenting  vat 
itself,  the  whole  brew  is  transferred  to  large  wooden  pans,  the 
so-called  "clarifiers,"  where  in  a  layer  about  one  foot  deep  the 
fermentation  process  is  carried  to  the  end.  Although  in  this  way, 
by  giving  the  mixture  a  large  surface,  the  rise  of  temperature  is 
checked,  the  fermentation  process  still  proceeds  with  considerable 
speed  on  account  of  the  ample  aeriation.  The  use  of  these 
"clarifiers"  is  a  characteristic  feature  in  the  manufacture  of  the 
California  steam  beer. 

The  yeast  of  the  steam  beer  has  accommodated  itself  to  these 
conditions  to  such  an  extent  that  it  can  no  longer  be  employed 
for  the  preparation  of  lager  beer,  while  lager-beer  yeast  may 
without  difficulty  be  used  for  the  manufacture  of  steam  beer. 
The  cells  of  the  typical  steam-beer  yeast  are  somewhat  smaller 
than  those  of  lager-beer  yeast. 

The  yeast  used  for  the  most  part  in  the  experiments  which  I 
am  about  to  describe  was  furnished  by  the  California  Brewing 
Company  of  San  Francisco.  I  am  highly  indebted  to  Mr.  G. 
Woehrle  of  this  firm,  who  very  kindly  supplied  me  with  the 
necessary  material  at  many  occasions. 

After  following  the  directions  of  Hill  (loc.  cit.)  and  Emmer- 
ling  (loc.  cit.)  in  every  detail,  I  arrived  at  the  conclusion,  after 
numerous  trials,  that  with  my  particular  material  (California 


160        University  of  California  Publications  in  Physiology  [Voi..  4 

steam-beer  yeast)  it  was  impossible  to  obtain  an  active  maltase 
prepartion  by  the  method  of  Hill.3 

The  following  are  only  a  few  of  the  many  negative  results. 
Two  mixtures  were  prepared,  each  containing  90  c.c  of  a  5  per 
cent  solution  of  pure  maltose  (Kahlbaum),  20  c.c.  yeast  extract 
(according  to  Hill),  and  1  c.c.  of  toluol.  The  solutions,  in  tightly 
stoppered  vessels,  were  placed  in  incubators,  one  at  a  constant 
temperature  of  30°  C.,  the  other  at  70°  C. 

From  time  to  time,  samples  of  10  c.c.  were  removed  with  a 
pipette  and  transferred  to  a  200  c.c.  graduated  flask.  After 
making  up  to  volume  with  distilled  water,  the  optical  rotation 
was  determined.  The  polariscope  used  was  a  Schmidt  and 
Haensch  triple  field  nicol-prism  instrument,  which  allowed 
readings  to  be  made  within  a  hundredth  of  a  degree.  Using  a 
400  m.m.  tube  and  white  light,  the  following  readings  were 
obtained. 

Time  in  hours  Polariscope  reading 

30°  C.  70°  C. 

0  1.10°  1.10° 

14  1.12°  1.10° 

42  1.10°  1.10° 

88  1.06°  1.04° 

162  1.10°  1.05° 

Part  of  the  yeast  extracted  was  placed  in  the  70°  incubator 
for  one  day.  A  whiteish  precipitate  had  formed  at  the  bottom. 
From  the  supernatant  clear  liquid,  after  cooling,  20  c.c.  were 

3  It  is  not  at  all  surprising  that,  in  working  with  living  material, 
methods  that  have  been  found  useful  for  a  certain  operation  in  one 
locality  may  not  be  successful  if  employed  at  a  different  locality.  Ex- 
periences of  that  sort  have  been  met  much  more  frequently  perhaps  than 
could  be  concluded  from  the  literature  alone.  The  lower  forms  of  life, 
such  as  yeasts  and  bacteria,  are  especially  sensitive  to  slight  variations 
of  external  conditions.  Such  changes  must  necessarily  affect  the  one-cell 
organism  much  more  deeply  in  its  whole  structure  and  organization  than 
they  would  influence  higher  forms,  where  those  variations  may  affect 
only  the  function  of  one  special  organ.  It  is  of  interest  in  this  connection 
that  with  yeast  material  of  this  locality  I  am  not  the  first  one  to  report 
a  complete  failure  of  a  method  that  is  generally  found  successful  in  other 
places.  Taylor  (A.  E.  Taylor,  On  Fermentation,  Univ.  Calif.  Publ.  Pathol., 
vol.  1,  p.  212)  after  many  unsuccessful  trials  to  obtain  by  the  Buchner 
method  (see  H.  Euler,  loc.  cit.,  p.  38)  an  active  zymase  preparation  from 
San  Francisco  yeast,  is  led  to  the  statement  that  "the  commercial 
Sticcharomyces  cerevisiae  of  this  city  is  worthless  for  the  preparation  of  a 
yeast  powder:  the  glycogen  content  is  high,  the  proteolytic  ferment  active, 
the  zymase  weak." 


19121  Birckner:  Yeast  Glucase  161 

tested  against  maltose  in  exactly  the  same  manner  as  described. 
The  result  was  the  following: 

Time  in  hours  Polariscope  reading 

30°  C.  70°  C. 

0  1.10°  1.10° 

14  1.10°  1.10° 

41  1.10°  1.09° 

71  1.07°  1.07° 

143  1.08°  1.07° 

Likewise  the  white  precipitate  alone  was  tested  against 
maltose  with  the  following  result : 

Time  in  hours  Polariscope  reading 

30°  C.  70°  C. 

0  1.31°  1.31° 

14  1.31°  1.31° 

41  1.30°  1.31° 

90  1.28°  1.28° 

In  all  three  cases,  it  is  seen,  there  was  practically  no  activity 
against  maltase. 

At  the  time  when  I  still  had  some  hope  of  having  maltase  in 
my  solution,  part  of  the  extract  wras  tried  on  a  strong  solution 
of  glucose,  on  which  maltase,  according  to  Croft  Hill  (loc.  cit.) 
and  Emmerling  (loc.  cit.),  exerts  a  synthetic  action,  yielding  a 
di-saccharide. 

I  noticed  very  peculiar  changes  to  take  place.  After  stand- 
ing in  the  70°  incubator  for  one  day,  the  sample  showed  a 
reddish  brown  coloration,  which,  after  two  or  three  days,  had 
changed  into  dark  crimson,  the  liquid,  which  was  still  perfectly 
clear,  having  acquired  an  aromatic  odor.  An  acid  reaction 
against  litmus  paper  wras  also  observed.  After  prolonged  stand- 
ing in  the  70°  incubator  in  tightly  stoppered  test  tubes,  the 
samples  turned  almost  black,  and  a  brownish  carbon-like  sub- 
stance settled  out  along  the  edges  of  the  glass  very  gradually. 
No  gas  formation  was  noticed. 

The  samples  which  had  been  kept  in  the  30°  incubator  under- 
went no  visible  changes  for  a  very  long  time,  after  which  they 
finally  became  faintly  yellow.  They,  too,  gradually  gave  an  acid 
reaction. 

The  fact  that  a  solution  of  glucose  alone  in  the  same  con- 


162        University  of  California  Publications  in  Physiology  [VOL.  4 

centration  did  not  undergo  any  similar  changes  could  be  easily 
ascertained.  Nor  did  either  maltose  or  the  ferment  extract,  or 
both  together,  show  any  such  colorations  for  at  least  two  weeks. 
It  was  therefore  natural  to  assume  that  a  catalytic  agent  of  some 
kind  was  bringing  about  these  changes  in  the  glucose-extract 
mixture. 

That  the  substance  which  causes  the  crimson  coloration  is 
actually  formed  at  the  temperature  of  70°  C.  and  not  simply  in 
the  time  during  which  the  mixture  is  gradually  being  heated  up 
to  this  point,  was  shown  by  placing  each  of  the  two  solutions 
(glucose  and  extract)  in  the  70°  incubator  separately  at  first, 
and  mixing  them  when  warm.  The  coloration  appeared  in  the 
same  way  as  before. 

On  transferring  some  of  the  colorless  samples,  which  had 
been  kept  in  the  30°  incubator  for  weeks,  to  the  70°  incubator, 
the  change  in  color  took  place  readily,  and  it  could  be  rendered 
twice  as  intense  by  boiling  the  sample  for  a  moment  just  before 
transferring  it.  The  respective  compound,  therefore,  had  pos- 
sibly been  formed  at  the  lower  temperature  too,  and  only 
assumed  that  different  color  at  the  elevated  temperature.  At 
any  rate,  it  seemed  important  to  know  what  was  the  nature  of 
the  substance  which  causes  these  characteristic  transformations 
with  such  regularity. 

I  was  at  first  inclined  to  think  that  really  a  synthetic  change 
from  glucose  into  isomaltose  had  taken  place,  an  assumption 
which  seemed  well  justified  with  regard  to  what  is  known  about 
the  properties  of  this  di-saccharide.4  Very  soon,  however,  I  could 
convince  myself  that  this  conclusion  was  erroneous. 

All  di-saccharides  are  known  to  have  a  higher  optical  rotation 
and  a  lower  copper  reducing  power  than  d-glucose.  In  §bme 
preliminary  experiments  I  had  already  noticed,  however,  that 
there  was  taking  place  in  my  mixtures  not  only  a  decrease  in 
reducing  power,  but  also  a  decrease  in  optical  activity.  These 
results  had  been  obtained  with  a  ferment  preparation  that  was 
more  than  two  months  old;  they  therefore  needed  confirmation. 
"With  a  fresh  preparation  the  result  was  largely  the  same,  as  we 
shall  see  presently. 


4  See  E.  v.  Lippmann,  loc.  cit.,  p.  1513. 


1912]  Birckner:  Yeast  Glucase  163 

To  160  c.c.  of  a  40  per  cent  solution  of  pure  glucose  (Kahl- 
baum),  40  c.c.  of  yeast  extract  and  a  few  drops  of  toluol  were 
added,  the  liquid  well  mixed,  and  measured  from  a  burette  into 
test  tubes  in  portions  of  exactly  10  c.c.  The  tubes  were  tightly 
stoppered  and  divided  into  two  sets.  One  set  was  placed  in  an 
incubator  at  30°  C.,  the  other  into  the  70°  incubator.  From 
time  to  time,  one  test  tube  of  each  set  was  taken  for  analysis,  its 
contents  being  washed  into  a  one-litre  graduated  flask.  After 
adding  10  c.c.  of  a  2.5  per  cent  solution  of  sodium  carbonate  to 
stop  the  reaction,  the  liquid  was  made  up  to  mark  with  distilled 
water,  filtered,  if  necessary,  and  an  aliquot  used  for  the  deter- 
mination of  the  reducing  power,  while  the  remainder  was 
available  for  the  polariscope  reading.  The  liquid  had  to  be 
used  in  such  a  high  dilution  on  account  of  the  coloration,  and 
because  it  is  not  advisable,  with  white  light,  to  obtain  readings 
that  would  exceed  the  value  of  three  degrees.5  The  white  light 
of  a  50-candle  power  globe  was  used  with  the  instrument,  as  I 
could  not  procure  a  sodium  light  of  sufficient  intensity,  The 
variations  in  temperature  were  slight,  and  their  influence  on 
the  reading  was  negligible  in  this  preliminary  work.  The  read- 
ings refer  to  a  tube  length  of  400  m.m. 

The  determinations  of  the  reducing  power  were  all  made  by 
the  method  of  Bertrand  (loc.  cit.).  In  the  tables  below,  which 
are  intended  to  give  an  approximate  idea  of  the  progress  of  the 
reaction,  only  the  number  of  cubic  centimeters  of  the  standard 
KMn04  solution  which  is  used  in  this  method  have  been  inserted. 
Each  cubic  centimeter  is  equivalent  to  8.72  mg.  Cu.  The 
mixture  at  the  beginning  was  composed  of  40  c.c.  yeast  extract, 
80  c.c.  glucose  (40%)  and  toluol.  (10  c.c.  were  made  up  to 
1  litre  for  each  determination.) 


Rotation 
Polariscope  reading 
30°  C.                 70°  C. 

Time 

in 
hours 

Reduction 
No.  of  c.c.  KMnO4  required 
30°  C.                      70°  C. 

0. 

,60 

0, 

.60 

0 

11, 

.62  c.c. 

11, 

62  c.c. 

0. 

,57 

0, 

,48 

64 

11 

,  70  c.c. 

11, 

55  c.c. 

0, 

,57 

0, 

,45 

110 

11 

.  65  c.c. 

11, 

,  35  c.c. 

0 

.53 

0, 

.42 

190 

11 

.58  c.c. 

11. 

25  c.c. 

0 

.54 

0 

.41 

254 

11 

.  65  c.c. 

11 

,15  c.c. 

5  See  Landolt-Long,  The  Optical  Eotating  Power  of  Organic  Substances 
(Easton,  Pa.,  The  Chemical  Publishing  Co.,  1902),  p.  417. 


164        University  of  California  Publications  in  Physiology  [VOL.  4 

The  yeast  extract,  as  already  mentioned,  gave  a  precipitate  in 
the  70°  incubator  if  kept  there  for  several  hours.  Both  frac- 
tions, the  clear  fluid  and  the  precipitate,  were  tested  separately 
for  their  action  on  glucose.  The  precipitate  showed  no  action 
whatever,  while  the  clear  liquid  had  preserved  its  fermentative 
qualities  almost  unweakened.  In  the  following  table  the  com- 
position of  the  mixture  was  exactly  the  same  as  in  the  preceding 
one,  except  that  for  the  ordinary  yeast  extract  this  clear  liquid, 
which  has  just  been  described,  was  substituted : 


Rotation 

Time 

Reduction 

Polariscope  reading 

in 

No.  of  c.c.  KMnO4  required 

30°  C. 

70°  C. 

hours 

30°  C. 

70°  C. 

0.55 

0.55 

0 

11.  63  c.c. 

11.  63  c.c. 

0.53 

0.48 

63% 

11.  58  c.c. 

11.  60  c.c. 

0.54 

0.42 

1*234 

11.  60  c.c. 

11.  35  c.c. 

From  both  these  tables  it  follows  clearly  that  there  is  no 
synthetic  process  going  on,  but  that  sugar  is  being  broken  down. 
On  these  figures  alone  we  could,  however,  not  base  a  reasonable 
interpretation  either  of  the  rate  of  the  reaction  or  of  its  products, 
for  the  following  reasons : 

Although  highly  diluted  for  the  analysis,  the  color  of  the 
liquid  deepens  to  such  an  extent  as  the  reaction  proceeds  that 
this  color  alone  causes  a  marked  depression  of  the  polariscope 
reading,  so  that  the  actual  loss  of  rotatory  power  is  probably 
not  nearly  so  great  as  would  appear  from  the  tables.  On  the 
other  hand,  the  fermentation  products  may  be  optically  active 
substances  themselves,  so  that  in  no  case  would  the  polariscope 
reading  give  an  accurate  idea  of  what  transformations  are  really 
taking  place.  Hence,  although  from  the  last  two  tables  it  seems 
as  if  the  fall  in  optical  activity  proceeds  much  faster  than  the 
fall  of  the  reducing  power,  we  can  not  at  all  be  sure  that  such 
is  really  the  case. 

The  destruction  of  glucose  may,  on  the  other  hand,  yield 
substances  which  themselves  have  a  high  reducing  power  against 
Fehling's  solution.  Hence  this  method  of  determination  might 
be  just  as  inaccurate  and  misleading  in  its  results  as  the  optical 
method. 


1912] 


Birckner:  Yeast  Glucase 


165 


I  therefore  had  no  accurate  means  of  measuring  the  progress 
of  the  reaction,  nor  was  there  much  chance  of  finding  out,  within 
a  short  time,  what  the  products  of  transformation  would  be. 
Moreover,  as  long  as  I  was  following  the  method  of  Hill,  I  only 
could  prepare  a  small  quantity  of  yeast  extract  at  a  time.  My 
particular  material  dried  only  very  slowly  in  the  vacuum  over 
sulphuric  acid;  and  had  to  be  spread  in  very  thin  layers,  thus 
giving  only  small  yields. 


Fortunately,  while  in  these  difficulties,  I  happened  to  gain  a 
new  aspect  of  the  problem  by  observing  that  the  ferment  showed 
a  certain  activity  against  hydroquinone,  hastening  its  oxydation 
to  quinone.  Hence  it  appeared  to  be  a  typical  oxydase.6  I 
therefore  decided  to  prepare  a  larger  quantity  of  it,  and  if  pos- 
sible to  find  a  somewhat  simpler  method  of  preparation. 

The  new  yeast  material  was  procured  from  a  larger  brewery 
this  time,  which  was  located  not  far  from  the  one  previously 
mentioned.  This  new  yeast  contained  slightly  less  bacteria  (only 
two  per  1000  yeast  cells)  than  that  of  the  California  Brewing 
Company  at  that  time.  Both  the  former  as  well  as  the  latter 
were,  however,  typical  steam-beer  yeasts. 

I  prepared  the  yeast  powder  in  three  different  ways : 

(1)  By  following  the  directions  of  Hill. 

(2)  By  treating  the  yeast  with  acetone  and  ether. 

(3)  By  treating  the  yeast  with  methyl  alcohol  and  ether. 
My  expectations  that  this  material  would  yield  the  ferment 

in  a  very  pure  and  active  condition  were  not  fulfilled.  In  fact 
I  lost  nearly  two  months'  time  in  preparing  the  three  different 
powders,  extracting  them,  and  testing  the  extracts  against 
different  solutions  of  sugar  and  hydroquinone  with  improved 
methods.  The  final  conclusion  which  I  had  to  draw  was  that  this 
yeast  does  not  contain  a  sufficient  amount  of  the  ferment  in 


o  Griiss  and  Issajew  had  already  described  the  presence  of  an  oxydase 
in  yeast  (Griiss,  Tiber  Oxydaseersehienungen  der  Hefe,  Woschenschr.  f. 
Brauerei,  vol.  18,  pp.  310,  335,  1901;  cf.  Kastle,  The  Oxydases,  etc.,  Hyg. 
Lab.  U.  S.  Pub.  Health  and  Marine-Hasp.  Serv.,  Bull.  59,  Washington,  1910; 
W.  Issajew,  liber  die  Hefe-oxydase,  Zeitschr.  f.  Physiol.  Chemie,  vol.  42, 
p.  132,  1904.). 


166        University  of  California  Publications  in  Physiology  [VOL.  4 

question,  to  be  used  with  advantage  for  the  purpose  of  its  study. 
Although  the  presence  of  the  ferment  in  the  extract  was  beyond 
doubt,  the  degree  of  activity  was  in  all  cases  only  a  small  fraction 
of  that  displayed  by  the  former  yeast  preparations. 

As  the  reasons  for  this  unexpected  behavior  I  finally  recog- 
nized the  following  facts : 

It  is  well  known  that,  as  a  rule,  ferment  preparations  are 
rapidly  destroyed  in  aqueous  solution  by  spontaneous  hydrolysis 
under  the  influence  of  antibodies  (proteolytic  ferments),  which 
are  mostly  quite  active  at  ordinary  temperature.  The  applica- 
tion of  low  temperature  usually  tends  to  increase  the  stability 
of  the  ferment,  by  depressing  this  autohydrolytic  process. 

Accordingly,  I  had  placed  some  of  my  clear  extracts  on  ice 
at  several  occasions.  After  a  few  days  these  samples  showed 
strong  turbidity  and  a  decreased  fermentative  activity.  On  the 
other  hand,  one  of  my  first  preparations,  which  had  been  stand- 
ing together  with  toluol  in  broad  daylight  in  a  rather  warm 
place  for  fully  six  months,  had  remained  clear  and  had  preserved 
almost  its  full  activity  against  glucose.  The  stability  of  this 
particular  ferment  is  therefore  not  favored  by  low  temperature. 
Now,  the  reason  that  the  yeast  of  the  California  Brewing  Com- 
pany contains  this  ferment  in  such  appreciable  amounts,  is  very 
likely  to  be  sought  in  the  fact  that  this  brewery  does  not  possess 
an  ice-plant.  During  the  time  when  the  yeast  is  not  in  action, 
it  is  hung  up  in  sacks  in  the  fermentation  room,  which  occupies 
the  second  floor  of  the  building,  being  well  aeriated  but  not 
artificially  cooled.  In  the  larger  establishment,  however,  from 
which  I  obtained  my  last  working  material,  the  steam-beer  yeast, 
in  the  same  way  as  the  lager-beer  yeast,  is  kept  in  a  cellar  cooled 
by  ice.  As  there  are  no  differences  in  the  treatment  of  the  jteast 
during  its  fermenting  action  at  the  two  different  places,  it  is 
most  probably  the  use  of  this  refrigerator  to  which  my  ill  success 
was  due  with  the  last  preparations.  It  is  obvious,  from  what 
has  been  said,  wyhy  this  yeast  does  not  develop  this  particular 
ferment  but  in  very  small  amounts. 

Naturally  under  these  circumstances  I  had  to  return  to  the 
use  of  the  original  yeast  material.  The  first  aim,  as  already 
stated,  was  to  find  out  which  way  of  preparing  the  yeast  powder 


Birckner:  Yeast  Glucase  167 

would  be  the  one  that  has  the  least  obnoxious  effects  on  the 
ferment. 

The  experiments  with  those  weak  extracts,  mentioned  in  the 
preceding  paragraph,  unsatisfactory  as  they  were  on  the  whole, 
had  shown  with  fair  certainty  that  the  acetone  yeast  as  well  as 
the  methyl  alcohol  yeast  (Dauerhefe)  on  extracting  yield  fer- 
ment preparations  which  are  in  every  respect  equivalent,  if  not 
superior,  to  those  prepared  by  Hill's  method.  They  had  shown, 
moreover,  that  the  ferment  in  question  is  most  likely  not 
zymase,  as  zymase  is  rapidly  destroyed  by  methyl  alcohol.7  They 
had  finally  shown  another  not  unimportant  fact.  One  of  the 
powders,  namely  the  CH3OH  powder,  happened  to  be  already 
neutral  to  litmus  paper  in  its  aqueous  suspension,  while  the 
others,  being  acid,  were  neutralized  with  dilute  Na2C03,  when 
first  brought  into  contact  with  water.  Now  I  observed  that  the 
extract,  which  was  already  neutral  by  itself,  not  only  gave  prac- 
tically no  precipitate  at  70°  C.,  but  together  with  glucose,  the 
mixture  kept  much  clearer  in  appearance  than  in  the  samples 
which  contained  the  neutralized  extracts,  notwithstanding  the 
coloration  process.  As,  besides,  the  ferment  did  not  seem  to  be 
very  sensitive  to  a  slightly  acid  reaction  of  the  medium  (it  was 
naturally  formed  in  a  medium  of  pronounced  acidity),  I  have 
in  all  subsequent  cases  avoided  the  addition  of  alkali.  By 
thoroughly  washing  the  yeast  all  the  acid  can  be  removed,  so 
that  the  resulting  powder  is,  on  extracting,  practically  neutral 
to  litmus. 

Above  all,  however,  the  tedious  method  of  Hill  (loc.  cit.) 
could  now  be  discarded  entirely.  A  new  batch  of  yeast  was  all 
prepared  as  "Dauerhefe."  Three  different  fixing  agents  were 
tried  once  more,  however,  in  order  to  determine  definitely  which 
of  them  could  finally  be  used  with  the  greatest  advantage. 

The  yeast  was  at  first  washed  three  times  with  tap  water  by 
decantation  in  glass  vessels,  after  thorough  stirring  at  each 
application  of  water.  Then  the  same  was  repeated  twice  with 
distilled  water,  Avhereupon  the  thick  suspension  was  transferred 
to  a  large  Buchner  funnel,  and  the  liquid  removed  by  suction. 


7  E.  Buehner,  H.  Buchner  und  M.  Hahn,  Die  Zymasegarung,  1903 ;   cf . 
W.  Zaleski,  Biochem.  Zeitschr.,  vol.  31,  p.  195,  1911. 


168        University  of  California  Publications  in  Physiology  [VOL.  4 

Now  the  compressed  yeast  was  divided  into  three  portions.  The 
first  was  treated  with  pure  acetone,  stirred  thoroughly,  and  the 
suspension  poured  on  a  Buchner  funnel,  and  the  acetone  removed 
with  the  air-pump  as  fast  as  possible.  The  wet  mass  was  now 
replaced  in  a  dish,  stirred  with  pure  ether,  and  the  latter  again 
removed  by  suction.  Then  acetone  was  applied  for  a  second  time, 
followed  again  by  ether,  and  the  resulting  powder  was  dried  in 
the  air.  The  process  is  essentially  the  same  as  that  employed 
by  Herzog  and  Saladin.8  Exactly  the  same  procedure  was 
observed  with  a  second  portion  of  the  compressed  yeast,  except 
that  instead  of  acetone  strong  methyl  alcohol  was  used;  and 
likewise  with  the  third  portion,  for  which  ethyl  alcohol  was  the 
fixing  agent.  The  dried  powder  of  each  portion  was  kept  in 
a  glass-stoppered  vessel,  and  part  of  it  removed  and  ground  in  a 
mortar  whenever  an  extraction  was  to  be  made.  The  two  alcohol 
preparations  represent  white,  dust-like  powders,  while  the 
acetone  preparation  is  heavier,  and  of  a  somewhat  sandy 
character. 

On  extraction  with  water,  all  except  the  CH3OH  preparation, 
which  reacts  slightly  acid,  are  neutral  to  litmus  paper.  The 
CH3OH  suspension  settles  quickly  and  is  easy  to  filter,  while  the 
other  two,  especially  the  acetone  preparation,  settle  very  slowly, 
and  are  very  difficult  to  filter. 

All  three  preparations,  after  passing  through  a  Chamberland 
filter  by  means  of  a  pressure  pump,  were  not  used  directly,  but 
were  submitted  to  a  process  of  repeated  precipitations  with 
alcohol  and  re-dissolutions  in  water,  a  process  which  I  shall 
describe  later  in  the  section  on  purification  methods.  The  fer- 
ment was  finally  obtained  in  the  form  of  a  crisp  powder  which 
was  partly  dried  in  the  vacuum  after  washing  with  alcohol, 
partly  after  additional  washing  with  ether.  To  my  knowledge 
ether  has  never  been  used  so  far  in  this  connection.  But  as  the 
evaporation  of  the  alcohol  is  a  slow  process,  even  in  the  vacuum 
over  sulphuric  acid,  I  tried  the  possible  effect  that  washing  with 
ether  might  have  on  the  ferment. 


s  B.  O.  Herzog  und  O.  Saladin,  tiber  die  Veranderungen  der  fermenta- 
tiven  Eigenschaften  welche  die  Hefezellen  bei  der  Abtotung  mit  Aceton 
erleideu,  Zeitschr.  f.  Physiol.  Chemie,  vol.  73,  p.  263,  1911. 


1912] 


Birckner:  Yeast  Glucase 


169 


Finally,  a  small  portion  of  each  of  the  three  raw  extracts 
was  slowly  boiled  for  six  minutes  with  animal  charcoal9  and 
filtered  before  being  precipitated. 

All  these  different  portions  were  now  tested  for  their  action 
on  hydroquinone.  The  apparatus  employed  for  this  purpose  was 
the  same,  in  principle,  as  that  described  by  Euler.10  I  have, 
however,  applied  a  few  alterations  which,  I  believe,  assure  a 
higher  accuracy  of  the  readings  without  complicating  the  method 
as  such.  Thus,  instead  of  a  burette  I  used  a  thin  glass  tube  of 
100  cm.  length,  to  which  I  attached  a  measuring  scale,  in  order 
to  secure  a  smaller  cross-section.  The  capacity  of  the  full  length 
of  the  tube  (100cm.)  was  only  17.72  c.c.  As  I  did  not  have  a 
vessel  of  the  kind  described  by  Euler  (loc.  cit.)  I  used  an 
ordinary  wide-mouth  bottle  of  rather  strong  glass,  having  an 
opening  of  3.7  cm.  in  diameter  and  a  capacity  of  about  500  c.c. 
Inside  of  this  bottle  was  first  placed  a  small  weighing  flask  into 
which  the  ferment  solution  was  measured  with  a  pipette.  Then 
the  respective  amount  of  hydroquinone  was  introduced  into  the 
larger  bottle,  and  the  latter  closed  with  a  tightly  fitting  rubber 
stopper  which  contained  two  glass  tubes,  one  connecting  with 
the  gasometer,  the  other  provided  with  a  glass  stop-cock,  as 
outlet  for  the  air.  After  the  bottle  had  been  filled  with  oxygen 
and  the  stop-cock  closed,  the  level  of  the  mercury  was  read  on 
the  scale  (bringing  the  mercury  in  both  branches  of  the  glass 
tube  to  the  same  level),  then  the  small  weighing  flask  tipped 
over,  and  the  bottle  put  on  the  shaking  machine,  which  was 
already  in  motion.  The  motor  was  arranged  so  as  to  give  the 
shaker  just  about  ninety  movements  a  minute,  which  was  found 
sufficient  to  accelerate  the  reaction  notably.  A  reading  was  taken 
every  minute  for  half  an  hour.  The  temperature  was  18°  C.  in 
all  experiments.  The  total  amount  of  liquid  in  each  case  was 
50  c.c.  and  it  was  always  0.2  normal  with  regard  to  hydroquinone. 
Equal  amounts  of  the  respective  ferment  solutions  of  equal 
strength  (0.2  g.  purified  ferment  powder  for  each  case)  were 


9  Specially  purified  with  HC1;  see  Euler,  as  below. 

10  H.  Euler  and  I.  Bolin,  Zur  Kenntnis  biologiseh  wichtiger  Oxydationen, 
Zeitschr.  f.  Physiol.  Cliemie,  vol.  57,  p.  80,  1908;  tiber  die  chemische  Zusam- 
mensetzung  und  die  biologisehe  Eolle  einer  Oxydase,  Zeitschr.  f.  PhysiJc. 
Cliemie,  Arrhenius  Jubelband  (69),  p.  187,  1909. 


170        University  of  California  Publications  in  Physiology  [VOL.  4 

employed  in  all  experiments,  so  as  to  make  possible  an  exact 
comparison  between  the  different  preparations.  The  amount  of 
oxygen  absorbed,  as  expressed  by  the  rising  of  the  mercury  in 
the  tube,  gave  a  measure  of  the  respective  rate  of  oxydation. 
"What  interests  us  here  is  chiefly  the  total  amount  of  oxygen 
absorbed  in  half  an  hour  by  the  hydroquinone  solution  under 
the  influence  of  the  different  ferment  preparations.  The  follow- 
ing table  expresses  these  amounts  as  measured  by  the  respective 
total  differences  in  centimeters  in  the  height  of  the  mercury 
level  in  the  tube.  The  corresponding  average  oxygen  absorption 
of  50  c.c.  of  a  0.2  normal  solution  of  hydroquinone  alone  was 
25  cm.  on  the  scale,  corresponding  to  4.43  c.c.  of  oxygen. 

Previous  treatment  of  Nature   of  fixing   agent : 

ferment  extract  Methyl  alcohol  Ethyl  alcohol  Acetone 

Final  washing  with  alcohol  42.2cm.  54.0cm.  55.0cm. 

Final  washing  with  ether  47.8cm.  52.0cm.  63.0cm. 

Boiled  for  6  minutes  16.4cm.  26.0cm.  24.4cm. 

For  all  three  preparations  the  influence  of  the  ferment  was 
therefore  readily  noticeable.  The  process  under  the  influence  of 
its  action  proceeded  with  at  least  twice  its  original  velocity.  No 
alkali  was  added  in  any  case.  The  washing  with  ether,  it  is 
noticed,  had  a  favorable  rather  than  an  obnoxious  effect  on  the 
ferment.  Boiling  for  six  minutes  had  destroyed  the  fermenta- 
tive power  entirely.  Compared  with  the  others,  the  acetone 
preparation  proved  to  be  the  most  active  against  hydroquinone. 

I  may  add  that  in  some  qualitative  experiments  I  convinced 
myself  that  the  addition  of  a  trace  of  MnS04  greatly  accelerated 
the  action  of  the  ferment  on  hydroquinone,  and  that  it  also 
accelerated  appreciably  its  action  on  glucose.  Hence  the*fer- 
ment  in  this  respect  resembled  Bertrand's  laccase.11 

On  trying  the  three  different  preparations  on  glucose,  I  found 
that  their  relative  activity  against  this  substance  was  not  simply 


n  G.  Bertrand,  Sur  le  pouvoir  oxydant  des  sels  manganeux  et  sur  la 
constitution  chimique  de  la  laccase,  Bull.  soc.  chim.  (3),  vol.  17,  p.  753, 
1897;  Sur  1 'action  oxydante  des  sels  manganeux  et  sur  la  constitution 
chimique  des  oxydases,  Compt.  rend.,  vol.  124,  p.  1355,  1897;  Sur  1 'inter- 
vention du  manganese  dans  les  oxydations  provoquees  par  la  laccase,  ibid., 
p.  1032. 


1912]  Birckner:  Yeast  Glucase  171 

in  parallel  with  their  respective  activity  on  hydroquinone.12 
While  against  hydroquinone  the  extract  of  the  acetone  powder 
caused  the  strongest  reaction,  this  extract  was  the  weakest  when 
acting  on  glucose,  judging  from  the  coloration.  Both  alcohol 
preparations  acted  much  stronger  in  the  latter  case.  But  ethyl 
alcohol  seemed  to  be  even  slightly  in  advance  of  the  methyl 
alcohol  preparation.  This  result,  in  consequence,  furnished  the 
standard  method  for  the  preparation  of  the*yeast  powder.  The 
following  is  the  way  in  which  I  proceed. 

METHOD  OF  PREPARING  THE  YEAST  POWDER 

The  yeast,  after  removing  from  the  clarifiers  (see  above)  is 
allowed  to  drip  in  the  brewery  for  about  two  days,  so  as  to 
become  fairly  dry.  About  fifty  pounds  may  now  be  taken  to  the 
laboratory.  The  mass  is  at  once  distributed  in  several  large 
glass  tanks,  so  as  to  fill  not  more  than  one-fifth  of  their  content. 
Then  while  stirring,13  a  stream  of  tap  water  is  turned  in,  and  the 
vessel  filled  to  the  edge.  After  the  mass  has  settled,  the  liquid  is 
siphoned  off,  and  new  water  added  while  stirring.  This  is 
repeated  a  third  time  with  tap  water,  and,  after  this,  twice  with 
distilled  water.  Great  care  should  be  exercised  in  this  work  in 
order  to  remove  all  the  acid  and  to  secure  a  neutral  reaction  of 
the  resulting  powder.  After  the  last  washing,  the  yeast  settle- 
ment is  poured  on  big  porcelain  funnels  (Buchner  form,  the 
bottom  of  which  consists  of  porous  clay),  and  the  liquid  drained 
off  by  suction.  The  sticky  compressed  mass  is  now,  in  portions, 
placed  in  a  large  porcelain  dish  with  absolute  alcohol  and  stirred 
(best  with  a  clean  hand).  The  suspension  is  poured  back  on  the 
funnel  and  the  alcohol  removed  by  suction.  The  mass  is  now 
again  placed  in  a  dish,  and  treated  in  the  same  way  with  ether. 
The  process  is  then  repeated,  once  with  alcohol  and  once  with 
ether,  care  being  taken  that  the  substance  does  not  stay  in  con- 
tact with  these  liquids  longer  than  necessary.  After  the  second 


12  This  is  in  agreement  with  Euler's  remark,  concerning  the  nomen- 
clature of  the  glucolytic  ferments,  to  which  I  have  referred  in  part  I, 
page  154. 

!3  I  usually  use  the  fingers  of  one  hand  (well  cleaned)  as  the  most 
effective  means  of  crushing  all  the  small  clods  of  yeast. 


172        University  of  California  Publications  in  Physiology  [VOL.  4 

treatment  with  ether,  the  whitish  mass  is  spread  on  filter  paper 
until  dry.  The  powder  is  to  be  kept  in  closed  vessels  in  a  warm 
and  dry  place. 

METHOD  OP  EXTRACTING  THE  FERMENT 

The  method  finally  adopted  for  getting  an  active  ferment 
preparation  is  the  following: 

A  portion  of  the  yeast  powder  is  ground  in  a  mortar  to  a  fine 
dust.  A  weighed  portion  of  this  is  placed  in  a  bottle  and 
thoroughly  shaken  with  ten  times  its  weight  of  distilled  water 
and  about  0.5  per  cent  of  toluol.  The  bottle  is  stoppered  and 
kept  at  a  temperature  of  between  30°  and  40°  C.,  or  at  room 
temperature.  At  the  end  of  thirty-six  hours  the  bottle  is  trans- 
ferred to  the  70°  incubator  for  another  thirty-six  hours.  During 
this  whole  period  of  extraction  the  bottle  is  shaken  from  time  to 
time.  At  the  end  of  the  second  thirty-six  hours  the  contents  of 
the  bottle  are  filtered,  at  first  through  paper,  and  then  through 
a  small  porcelain  funnel  with  clay  bottom,  using  suction.  The 
dark  yellow,  strongly  opalescent  filtrate  contains  the  ferment. 
It  may  be  kept  in  solution  indefinitely,  if  saturated  with  toluol, 
and  placed  at  room  temperature.  No  precipitation  will  occur. 
Any  addition  of  water,  however,  during  or  after  the  filtration 
process,  should  be  avoided,  as  a  strong  autodigestion  of  the 
extract  may  set  in  on  dilution. 

ATTEMPTS  AT  FURTHER  PURIFICATION 

Although  the  raw  extract  showed  a  distinct  activity  on  both 
glucose  and  hydroquinone,  the  progress  of  the  reaction  on 
glucose,  to  judge  from  the  coloration,  seemed  to  be  rather  slew, 
considering  the  usual  rapidity  of  fermentative  accelerations.  I 
had  noticed  at  the  beginning  that  the  yeast  gave  a  strong  reaction 
with  iodine,  indicating  great  richness  in  glycogen.  Apparently 
the  extract  contained  also  a  good  many  gummy  substances;  and 
it  seemed  not  improbable  that  by  using  some  kind  of  a  purifica- 
tion process,  I  might  be  able  to  eliminate  part  of  these  substances. 

At  first  the  method  of  repeatedly  precipitating  the  ferment 
with  alcohol  and  redissolving  in  water  was  tried,  a  method  which 


1912]  Birckner:  Yeast  Glucase  173 

has  frequently  been  used,  especially  when  working  with  oxydases. 
I  followed  closely  the  method  described  by  Euler14  for  the 
Medicago  laccase.  The  freshly  prepared  ferment  extract  was 
poured  into  three  times  its  volume  of  98  per  cent  alcohol,  the 
precipitate  collected  on  a  hardened  filter  and  dried  in  the 
vacuum  over  sulphuric  acid.  When  dry,  it  formed  a  brownish, 
gluey  mass.  This  was  now  redissolved  in  water,  filtered,15  and 
precipitated  again  by  pouring  the  filtrate  slowly  into  three  times 
its  volume  of  absolute  alcohol.  The  precipitate  was  again 
collected  on  a  filter,  washed  with  alcohol  and  dried.  This 
manipulation  was  repeated  a  third  time.  But  even  after  this 
the  resulting  substance  was  not  a  white  powder,  as  Euler 's 
Medicago  laccase,  but  a  very  brittle  porcelain-like  mass,  showing 
that  there  are  still  a  good  many  gummy  substances  contained  in 
it.  The  process  can  be  somewhat  improved  by  using  ether  for 
washing  the  precipitate,  in  which  case  the  operation  of  drying  is 
hastened  considerably,  while  the  ether,  as  can  be  seen  from  the 
table  on  page  170  above,  has  no  injurious  effect  on  the  ferment. 
The  final  purification  product,  if  dissolved  in  water  and 
filtered,  is  a  colorless,  opalescent  fluid,  which  contains  the  fer- 
ment. It  was  with  preparations  of  this  kind  that  the  hydro- 
quinone  experiments16  were  carried  out.  Later  on,  I  observed, 
however,  that  against  glucose  these  preparations  showed  far  less 
activity  than  the  original  raw  extracts. 

Another  method  of  purification  was  tried  in  the  following : 
A.  Wurtz17  in  preparing  the  ferment  papain  from  Carica 
Papaya,  had  found  that  this  ferment,  although  a  protein  in 
character,  is  not  precipitated  by  basic  lead  acetate.  He  made  use 
of  this  observation  in  purifying  his  ferment  by  precipitating 
most  of  the  impurities  with  lead.  He  actually  could  obtain  from 
his  lead  filtrate  a  very  active  ferment  preparation.  The  same 
method  was  used  shortly  afterwards  by  0.  Loew18  for  the 


i*  Zeitschr.  f.  Physilc.  Chemie,  vol.  69,  p.  190,  1909. 

is  There  is  always  a  part  left  which  does  not  redissolve,  upon  which 
fact  the  purification  process  is  based. 

IB  See  above,  p.  170. 

IT  A.  Wurtz,  Sur  la  Papaine,  Contribution  a  1  'histoire  des  ferments 
solubles,  Compt.  rend.,  vol.  90,  p.  1379,  1880. 

is  Oscar  Loew,  "fiber  die  chemische  Natur  der  ungeformten  Fermente, 
Pfliiger's  Archiv.  f.  ges.  Physiol,  vol.  27,  p.  203,  1882. 


174        University  of  California  Publications  in  Physiology  [VOL.  4 

purification  of  diastase,  apparently  with  good  success.  Euler,19 
in  referring  to  Loew's  work,  misrepresents  this  method  by  stating 
that  Loew  had  set  free  his  ferment  from  the  lead  precipitate. 
At  any  rate,  it  seemed  worth  while  to  try  this  convenient  method 
with  my  ferment. 

I  followed  closely  the  directions  given  in  Loew's  article.  The 
filtrate  of  the  lead  precipitate,  however,  after  freeing  from  lead, 
did  not  give  any  precipitation  with  alcohol  in  my  case.  Nor 
could  a  precipitation  be  obtained  with  FeCl3.20  Therefore  the 
ferment  in  my  case  did  not  resist  precipitation  by  lead. 

The  lead  precipitate  was  now  suspended  in  a  small  amount  of 
water,  the  lead  taken  out  by  H2S,  and  the  lead  sulphide  filtered 
off.  The  filtrate  gave  a  very  small  precipitate  with  alcohol, 
which  on  dissolving  in  water  showed  no  fermentative  activity 
whatever.  Hence  this  method  was  of  no  advantage  for  my 
purpose. 

The  method  used  by  Frankel  und  Hamburg21  for  purifying 
diastase,  although  based  on  good  principles,  is  very  tedious,  and 
has  not  yet  been  tried  with  my  ferment.  Besides,  I  notice  that 
the  results  of  these  authors  could  not  be  confirmed  by  a  recent 
investigator.22 

PROPERTIES  OF  THE  YEAST  EXTRACT 

Besides  having  a  characteristic  action  on  glucose,  the  ferment 
extract  has  some  other  properties  which  deserve  attention.  In 
the  first  place  let  us  consider  its  qualities  as  an  oxydase.  If  a 
small  portion  of  the  extract  or  of  the  purified  powder  (alcohol 
purification)  is  added  to  a  dilute  solution  of  hydroquinone,  and 
the  mixture  shaken,  it  turns  red  after  a  few  minutes'  standing. 
A  similar  action  takes  place  with  pyrogallol,  the  mixture  turning 
yellow  on  short  standing.  No  color  change  occurs  with  guajacol, 


19  H.  Euler,  Allgemeine  Chemie  der  Enzyme,  p.  13,  1910. 

20  See  W.  Lob  und  Pulvermacher,  Biochem.  Zeitschr.,  vol.  29,  p.  316. 

21  S.  Frankel  und  M.  Hamburg,  Uber  Diastase.     1:  Versuche  zur  Her- 
stellung  von  Keindiatase  und  deren  Eigenschaften,  Hofmeister's  Beitrdge 
zur  chem.  Pliysiol.  u.  Path.,  vol.  8,  p.  389. 

22  F.  Miinter,  tiber  Enzyme  2te  Mitt.,  Landw.  Jahrbiicher,  vol.  39,  Erg. 
Bd.  3,  p.  298,  1910;  cf.  Zentralbl.  f.  Biochemie  und  Biophysik.,  vol.  11,  p. 
185,  1910-1911. 


1912  ]  BircJcner:  Yeast  Glucase  175 

nor  with  tincture  of  guajacum,  not  even  after  adding  some 
hydrogen  peroxyde.  In  this  respect  the  ferment  resembles 
Euler's  Medicago  laccase  (loc.  cit.}.  As  we  have  already  seen 
before,23  it  also  resembles  Bertrand's  Rims  laccase  (loc.  cit.)  in 
being  accelerated  in  its  action  by  manganese  salts. 

The  indophenol  test  (Rohmann-Spitzer's  reagent)24  was 
negative;  the  same  wras  also  the  case  with  Tollens'25  orcin 
reaction  and  Goldschmidt 's  test.26  Tollens'  reaction  with 
naphtoresorcin  was  also  negative.  No  coloration  occurred  with 
a-napthol,  nor  wTith  tannic  acid,  the  latter,  however,  giving  a 
precipitate.  Molisch's  test27  with  a-naphtol,  as  well  as  Neuberg's 
pyrrol  reaction,28  was  distinctly  positive,  even  with  the  prepara- 
tion which  had  been  precipitated  with  alcohol,  and  redissolved 
for  three  times.  The  carbohydrate  group  is  therefore  possibly 
in  firm  combination  with  the  ferment  molecule  and  may  be 
regarded  as  one  of  its  essential  constituents.  Possibly  this  factor 
is  of  significance  for  determining  the  specifity  of  the  ferment 
(see  Armstrong,  loc.  cit.,  p.  58). 

The  ferment  in  aqueous  solution  is  slightly  dextrorotatory; 
it  does  not  reduce  Fehling's  solution.  It  causes  no  color  change 
on  being  added  to  a  solution  of  tyrosin,  and  therefore  contains 
no  tyrosinase.  It  has  a  slight  action  on  sodium  lactate,  liberat- 
ing an  acid,  without  the  formation  of  gas.  The  ferment  gives  all 
protein  reactions ;  boiling  for  one  hour  does  not  cause  any  pre- 
cipitation ;  it  is  precipitated  by  alcohol  and  ether. 

The  yeast  extract  contains  evidently  at  least  two  different 
ferments : 


23  See  above,  p.  170. 

24  F.  Kohmann  und  W.  Spitzer,  tiber  Oxydationswirkungen  tierischer 
Gewebe,  Ber.  Deutschr.  diem.  Ges.,  vol.  28,  p.  567,  1895. 

25  Loc.  cit. 

20  G.    Goldschmidt,   Eine   neue   Eeaction   auf   Glucuronsaure,    Zeitschr. 
f.  Physiol.  Chemie,  vol.  65,  p.  390,  1910. 

27  H.   Molisch,   Zwei   neue   Zuckerreaktionen,   Monatschefte   f.   Chemie, 
vol.  7,  p.  198,  1886. 

28  C.    Neuberg,   tiber   den    Nachweis    der    Bernsteinsaure,    Zeitschr.    f. 
Physiol.  Chemie,  vol.   31,  p.  574,  1900;   Zur  Kenntnis  der  Pyrrolreaktion, 
Chem.  Centralbl.,  vol.  2,  p.  1435,  1904. 


176        University  of  California  Publications  in  Physiology  [VOL.  4 

1.  An  oxydase,   active  against  poly  phenols,   which  is 
inactivated  by  boiling. 

2.  A  glucolytic  ferment  which  is  not  destroyed  by  heat, 
not  even  by  boiling  for  fifteen  minutes  in  a  pressure  flask. 

The  latter  ferment  deserves  particular  attention. 

STUDIES  ON  THE  PRODUCTS  OF  GLUCOSE  FERMENTATION 

Numerous  attempts  have  been  made  to  obtain  some  knowledge 
about  the  substance  into  which  glucose  is  transformed  under  the 
influence  of  this  ferment. 

If  glucose-ferment  mixtures  are  filled  into  Schrotter's  fer- 
mentation bulbs,  and  placed  in  the  70°  incubator,  the  progress 
of  the  reaction  can  be  observed  very  plainly.  The  liquid,  if  con- 
taining a  fairly  active  extract,  became  distinctly  acid  within  a 
few  hours,  and  by  and  by  the  coloration  took  place.  No  gas 
formation  was  observed  in  any  case.  The  iodoform  reaction  for 
alcohol  was  negative,  but  with  Pasteur's  droplet  test,  if  applied 
in  the  manner  as  recently  described  by  Klocker,29  the  presence 
of  traces  of  alcohol,  or  of  a  similar  substance,  wras  ascertained. 

The  sugar  is  therefore  mainly  transformed  into  acids.  The 
access  of  air  causes  the  reaction  to  proceed  slightly  faster,  but 
it  is  not  at  all  necessary,  as  I  have  convinced  myself  in  special 
experiments.  In  tightly  stoppered  flasks,  which  were  filled 
nearly  to  the  top,  leaving  only  about  1  c.c.  of  air  space,  the 
darkening  and  the  formation  of  the  dark  residue  occurred  almost 
as  readily  as  in  other  flasks  that  were  provided  only  with  cotton 
plugs.  As  a  matter  of  course,  toluol  was  added  to  all  cultures, 
although  the  high  temperature  alone  would  nearly  suppress 
bacterial  action. 

I  tried  several  ways  of  decolorizing  the  dark  mixture  with 
the  object  of  rendering  possible  the  application  of  the  polariscope 
method.  All  these  trials  were  unsuccessful  so  far.  In  the  mean- 
while I  have  found,  however,  that  a  complete  decolorization  of 
the  mixture  is  possible  by  means  of  a  combined  precipitation 


20  A.  Klocker,  Nachweis  kleiner  Alkoholmengen  in  garenden   Fliissig- 
keiten,  Centralbl.  f.  Bacteriologie  (n),  vol.  31,  p.  108,  1911. 


1912]  Birckner:  Yeast  Glucase  177 

with  mercuric  acetate  and  phosphotungstic  acid  in  the  manner 
recently  recommended  by  Neuberg.30 

All  color  reactions  with  this  mixture  naturally  had  to  be 
unreliable,  partly  in  direct  consequence  of  the  color,  partly  on 
account  of  the  many  substances  present.  Tollens'  orcin  reaction 
alone  indicated  with  some  certainty  the  presence  of  pentoses. 

The  dark  mixture,  if  added  to  Fehling's  solution,  reduced  it 
rapidly  in  the  cold.  As  according  to  Neuberg31  only  a  few  sugar 
derivatives  show  this  behavior,  namely  glucuronic  acid,  glycerose, 
dioxyacetone,  and  glucose,  I  was  led  to  believe,  also,  with 
regard  to  the  recent  results  of  Jolles,32  that  glucuronic  acid 
was  one  of  the  products.  As  at  that  time  I  did  not  have  at  hand 
the  reagents  to  make  sure  of  this  by  a  qualitative  color  reaction, 
I  started  a  distillation  process,  following  largely  the 'suggestions 
given  by  C.  Tollens.33  The  mixture,  after  filtering  and  cooling, 
was  precipitated  with  basic  lead  acetate  (no  ammonia  being 
added  on  account  of  the  glucose),  the  precipitate  washed  with 
water,  and  boiled  directly  with  HC1  (sp.  g.  1.060)  in  the  dis- 
tilling apparatus.  Part  of  the  filtrate  from  the  lead  precipitate 
was  also  distilled  over  with  HC1,  as  this  portion  would  contain 
any  pentose  that  might  possibly  have  been  formed.  If  the 
distillate  gave  the  furfurol  test  with  aniline  acetate  (see  C. 
Tollens,  loc.  cit.}  the  distillation  was  continued,  until  about 
450  c.c.  had  been  passed  over.  At  the  end,  a  solution  of  pure 
phloroglucin  (Kahlbaum)  in  HC1  was  added,  and  the  amount 
of  furfurol-phloroglucide  estimated  after  sixteen  hours'  stand- 
ing. In  no  case  did  the  lead  precipitate,  if  properly  washed, 
give  even  a  trace  of  this  substance,  while  the  filtrate  quite 
regularly  gave  a  fair  amount  of  phloroglucide.  This  filtrate,  of 
course,  still  contained  a  large  amount  of  unaltered  glucose,  and 
it  seemed  not  absolutely  certain  that  the  furfurol  was  really 


30  C.  Neuberg  und  M.  Ishida,  Die  Bestimmung  der  Zuckerarten  in 
Naturstoffen,  Biochem.  Zeitschr.,  vol.  37,  p.  142,  1911. 

si  C.  Neuberg,  Die  Physiologic  der  Pentosen  und  der  Glucuronsaure, 
Ergebn.  d.  Physiol.,  vol.  3i,  p.  387,  1904. 

32  Biochem.  Zeitschr.,  vol.  34,  p.  242. 

ss  C.  Tollens,  Quantitative  Bestimmung  der  Glueuronsaure  im  Urin  mit 
der  Furfurol  HC1.  Destinations  methode,  Zeitschr.  f.  Physiol.  Chemie,  vol. 
61,  p.  95,  1910. 


178        University  of  California  Publications  in  Physiology  [VOL.  4 

derived  from  pentose.34  To  investigate  this  point,  I  have  in  one 
case  treated  the  filtrate  of  the  lead  precipitate  with  H2S,  thus 
removing  all  the  lead.  The  H2S  was  driven  out  of  the  dark 
yellowish  filtrate  by  a  current  of  air,  and  the  liquid  fermented 
with  yeast.  The  solution  soon  turned  dark  crimson,  and, 
although  perfectly  clear,  assumed  a  strong  odor,  similar  almost 
to  that  of  indol  and  skatol.  The  tests  for  these  substances,  how- 
ever, gave  negative  results,  as  was  to  be  expected.  At  the  end 
of  the  fermentation  the  reducing  power  of  the  liquid  against 
Fehling's  solution  was  strongly  diminished,  and  the  Cu2O 
formed  had  a  peculiar  crimson  color.  That  the  reducing  agent 
in  this  case  was  not  glucose  was  ascertained  by  preparing  the 
osazone.  A  sample  of  the  liquid  was  boiled  in  the  water  bath 
for  one  hour  with  phenylhydrazine  hydrochlorate  and  sodium 
acetate.  It  was  then  allowed  to  cool  very  gradually.  On  exam- 
ining the  substance  under  the  microscope  after  several  hours,  I 
observed  only  traces  of  glucosazone  crystals,  while  the  liquid  was 
filled  with  masses  of  small  brownish  globule-like  crystals  of  an 
oily  appearance.  This  is  exactly  the  manner  in  which  the 
arabinosazone  is  described  by  von  Lippmann35  to  appear  when 
first  formed  in  the  presence  of  foreign  bodies.  In  fact,  011 
prolonged  standing,  a  solid  yellowish-brown  sediment  of  osazone 
crystals  separated  out.  On  examining,  the  long,  needle-shaped 
crystals  of  a  light  yellow  color  were  easily  seen  although  they 
were  densely  covered  with  a  rust-brown,  amorphous  substance. 
On  distilling  the  liqui'  over  with  HC1,  and  adding  phloroglucin 
to  the  distillate,  an  r  uple  precipitation  took  place.  Hence  the 
formation  of  pentose  seemed  sufficiently  assured;  while  any  for- 
mation of  glucuronic  acid  had  to  be  denied. 

Quantitative  studies  of  this  pentose  formation  are  in  progress. 

"With  one  of  the  early  digests,  which  contained  40  per  cent 
glucose,  I  made  an  ether  extraction,  shaking  the  mixture  with 
ether  in  a  separating  funnel,  and  using  fresh  ether  several  times. 
The  first  portions  of  this  extraction  were  unfortunately  lost. 


3-11 00  g.  glucose  may  yield  on  distillation  with  HC1  up  to  0.222  g. 

furfurol  (Stoklasa;  cf.  v.  Lippmann,  loc.  cit.,  p.  103). 

••<•"•  von  Lippmann,  Die  Chemie  der  Zuckerarten  (Braunschweig,  1904), 
p.  91. 


1912]  Birckner:  Yeast  Glucase  179 

With  the  dark  oily  syrup  at  the  bottom  and  ether  on  top,  the 
funnel  was  put  aside  for  a  long  time.  Finally,  on  testing,  I 
found  that  the  ether  had  become  strongly  acid.  Still  this  acid 
apparently  was  not  very  soluble  in  ether,  as  it  had  not  been 
removed  by  the  first  three  or  four  portions  of  ether.  Besides, 
when  I  tried  to  obtain  a  similar  extraction  from  an  ether  digest, 
which  did  not  contain  such  a  high  concentration  of  glucose,  the 
acid,  for  the  most  part,  stayed  in  the  aqueous  solution,  and  the 
ether  became  only  very  faintly  acid. 

The  strongly  acid  ether  fraction,  on  evaporating,  yielded  a 
yellowish,  oily  fluid  of  a  peculiar  odor.  This  liquid  was  taken 
up  in  water  and  carefully  distilled.  The  distillate  was  a  color- 
less, neutral  fluid  of  aldehydic  odor,  which  did  not  give  pre- 
cipitates with  CaCl,,  FeCl3,  or  alcohol.  It  gave,  however,  a  fine 
bluish-white  precipitate  with  AgN03,  which  turned  greyish 
brown  on  heating.  The  tests  for  aldehyde  gave  negative  results. 
This  substance  was  not  identified. 

The  acid  residue,  in  the  distilling  flask,  did  not  give  any  pre- 
cipitate except  with  lead  salts.  It  was  optically  inactive  and 
did  not  reduce  Fehling's  solution.  Tollens'  orcin  reaction,  as 
well  as  the  napthoresorcin  reaction  and  Goldschmidt 's  test  (loc. 
cit.)  was  negative.  Molisch's  test  with  a-naphtol  was  positive. 
On  evaporating  in  the  vacuum  over  sulphuric  acid,  the  acid 
liquid  did  not  crystallize  but  yielded  a  dark  syrup. 

If  to  this  syrup  a  concentrated  solution  of  potassium  acetate 
was  added,  crystals  were  formed  almost  immediately.  These 
granular  crystals  could  also  be  obtained  a  dilute  solution  in  a 
test  tube,  if  after  adding  potassium  acetate  the  walls  of  the  test 
tube  were  rubbed  with  a  glass  rod. 

I  obtained  just  enough  of  these  crystals  to  make  a  few  melt- 
ing point  determinations.  The  substance  did  not  melt  until 
above  300°  C.  I  prepared  the  acid  potassium  salts  of  saccharic 
acid  and  of  tartaric  acid  in  order  to  compare  their  respective 
melting  points  with  the  one  obtained.  The  melting  point  of  the 
saccharate  was  found  to  be  186?5  C.,  that  of  the  tartrate  about 
270°  C.,  while  the  melting  point  of  the  acid  potassium  oxalate, 
which  salt  is  rather  insoluble,  too,  was  found  to  lie  above  350°  C. 
I  presume  that  the  crystals  which  I  had  obtained  on  addition 


180        University  of  California  Publications  in  Physiology 


.4 


of  CH3COOK,  were  the  result  of  some  sort  of  secondary  trans- 
formation of  the  original  acid  into  oxalic  acid.  Primarily,  the 
ether  soluble  extract  did  certainly  not  contain  oxalic  acid,  as  no 
crystals  were  obtained  on  evaporation  and  as  the  calcium  pre- 
cipitate was  readily  soluble  in  acetic  acid  (see  table  below). 

If  the  free  acid  was  just  neutralized  with  ammonia,  several 
characteristic  reactions  could  be  obtained,  the  results  of  which 
I  have  arranged  in  the  following  table : 


Free  acid  Reagent 

No  precipitate  AgN03 

No  precipitate  FeCl3 

No  precipitate  CaCl2 

No  precipitate  Alcohol 


Ammonia   salt 

Yellow  precipitate;  soluble  in  the  cold  in 
NH3.  On  heating,  slight  reduction. 

White,  gelatinous  precipitate,  which  dis- 
solves slowly  in  N/10  HC1,  more  rapidly 
in  strong  HC1.  Also  dissolves  in  excess 
of  NH3  to  a  dark  yellow  solution  (of 
FeCl3  color). 

Thick  white  precipitate.  Insoluble  when 
heated,  and  in  cold  NaOH.  Dissolves  in 
N/10  HC1,  and  also  on  adding  a  few 
drops  of  glacial  acetic  acid. 

ll'Iiilc  precipitate.     Insoluble  in  ether. 


On  evaporating  the  solution  of  the  ammonia  salt,  a  greyish, 
crystalline  mass  of  caramel-like  odor  was  obtained.  Under  the 
microscope  it  appeared  to  consist  largely  of  rhombic  plates.  No 
exact  melting  point  could  be  obtained  with  this  residue.  Prob- 
ably it  was  not  a  single  compound,  but  a  mixture  of  several 
salts'.  Above  180°  C.  the  substance  in  the  capillary  tube  turned 
brownish,  but  it  had  not  melted  yet  at  a  temperature  of  250°  C. 

A  portion  of  the  dark  residue  of  the  ether  extraction  which 
I  mentioned  above,  was  diluted  with  water  and  carefully  dis- 
tilled; the  receiver  being  cooled  by  ice-water.  The  colorless 
clear  distillate  had  a  strong  odor  and  was  distinctly  acid  against 
litmus  paper.  It  gave  the  following  reactions : 

(a)   It  reduced  ammoniakal  silver  solution. 

(6)  After  acidifying  a  small  portion  with  H2SO4,  it  was 
shaken  in  a  separating  funnel  with  pure  chloroform. 
The  chloroform  was  drawn  off;  and  on  adding  to  it 
0.5  c.c.  of  Nessler's  reagent  (freshly  prepared),  a 
strong  yellow  coloration  of  the  latter  occurred  at  once. 


1912]  Birckner:  Yeast  Glucase  181 

(c)  A  crystal  of  resorcin  was  dissolved  in  a  few  drops  of  the 

liquid,  and  the  mixture  allowed  to  run  down  slowly 
along  the  side  of  a  test  tube  containing  concentrated 
sulphuric  acid.  At  the  zone  of  contact  a  bright,  orange 
red  color  developed  very  soon,  which  gradually  turned 
darker  red. 

(d)  The  distillate  gave  Schryver's30  formaldehyde  test  very 

plainly.  The  reaction  was  carried  out  in  the  follow- 
ing way : 

To  5  c.c.  of  the  liquid  were  added 

1  c.c.  of  a  1  per  cent  solution  of  phenylhydrazine 

chlorhydrate  (freshly  prepared  and  filtered), 
0.5  c.c.  of  a  freshly  prepared  5  per  cent  solution 

of  potassium  ferri-cyanide,  and 
2.5  c.c.  of  strong  HC1  (sp.  g.  1.19). 

The  mixture  showed  a  distinct,  though  not  very 
intense  red  coloration.  It  was  now  shaken  with  pure 
ether  in  a  small  separating  funnel,  and  the  lower  layer 
drawn  off.  To  the  ethereal  extraction  were  now  added 
1-2  c.c.  of  strong  HC1.  The  latter  assumed  almost 
immediately  a  very  intense  red  color,  indicating  the 
the  presence  of  formaldehyde. 

In  addition  to  this  substance,  the  distillate,  as  already  men- 
tioned, contained  a  volatile  acid  (possibly  formic  acid),  which 
however,  I  have  not  yet  identified. 

In  view  of  the  fact  that  W.  Lob  (loc.  cit.)  has  found  as  the 
products  of  electrolysis  of  glucose  polyoxyacids,  pentose,  and 
formaldehyde,  it  is  certainly  of  some  interest  that,  as  I  have 
shown,  a  ferment-like  substance  occurs  in  the  yeast  cell  which 
is  able  to  bring  about  (or  rather  to  accelerate)  the  cleavage  of 
the  glucose  molecule  into  essentially  the  same  products. 


3c  S.   B.   Schryver,   The  photochemical   formation   of   formaldehyde   in 
green  plants,  Eoy.  Soc.  Proc.,  82,  p.  226,  1910. 


182        University  of  California  Publications  in  Physiology  [VOL.  4 


CONCLUSIONS 

Summarizing  briefly  the  contents  of  this  article,  I  may  state 
the  following: 

1.  A  systematic  review  has  been  given  in  the  first  part  of 
the  paper  of  some  recent  advances  of  our  knowledge  of  glucose 
oxydations  and  cleavages,  both  outside  and  inside  of  the  organism. 

2.  In  the  second  part  of  this  article   a   ferment  has  been 
described  which  occurs  in  the  California  steam-beer  yeast  under 
certain  conditions,  and  which  has  the  property  of  accelerating 
the  decomposition  of  glucose  at  an  elevated  temperature. 

3.  This  new  ferment  is  not  identical  writh  zymase.     It  acts 
preferably  at  a  temperature  of  70°  C.     It  causes  no  gas  forma- 
tion and  yields  no  alcohol. 

4.  Its  action  on  glucose  at  70°  C.  manifests  itself  by  a  rapid 
darkening  of  the  mixture,   an   increase   in   acidity,   a   gradual 
formation  of  a  carbon-like  solid  settlement,  and  the  development 
of  an  odor  similar  to  that  of  caramel. 

5.  The    ferment    may   be    extracted    from    a    yeast    powder 
(Dauerhefe)  -which  is  best  obtained  by  killing  the  cells  with 
ethyl  alcohol. 

6.  From  a  wratery  extract  the  yeast  glucase  may  be  obtained 
and  purified  by  repeated  precipitation  with  alcohol;  but  this 
process  always  involves  a  weakening  of  the  ferment. 

7.  Yeast  glucase  is  very  stable  in  aqueous  solution,  if  kept  at 
room  temperature  under  sterile  conditions.     Boiling  does  not 
destroy  its  activity. 

8.  The  yeast  glucase  preparation  shows  activity  in  neutral  or 
acid  solution  against  glucose,  polyphenols,  and  lactates.    It  does 
not  contain  tyrosinase,  nor  does  it  act  as  a  peroxydase  against, 
glucose. 

9.  The  ferment  preparation  gives  a  strong  pyrrol  reaction 
(Neuberg). 

10.  Yeast  glucase  shows  some  relationship  to  the  oxydases, 
but  with  regard  to  its  main  function,  it  is  to  be  classed  together 
with    zymase    in    a    group    which   stands   separately    from    the 


Birckner:  Yeast  Glucase  183 


oxydases  and  the  hydrolytic  ferments,  and  to  which  Euler  has 
applied  the  name  "  Garungsenzyme  "  (see  p.  154). 

11.  The  transformation  products  of  glucose  resulting  from 
the  action  of  this  ferment  are  partly  acids,  none  of  which  has 
so  far  been  definitely  identified.  However,  among  the  cleavage 
products  of  the  sugar  the  presence  of  pentose  and  of  formalde- 
hyde could  be  ascertained. 

I  wish  to  express  my  thanks  to  Dr.  T.  Brailsford  Robertson 
for  his  valuable  counsel  and  continuous  interest  in  this  in- 
vestigation, and  also  to  Mr.  C.  B.  Bennett  for  kind  suggestions 
in  carrying  out  some  of  the  chemical  work. 

Transmitted  March  9,  1912. 


«;••• 


UNIVEBSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

21.  On  the  Local  Application  of  Solutions  of  Saline  Purgatives  to  the 

Peritoneal  Surfaces  of  the  Intestines,  by  John  Bruce  MacCallum. 
Pp.  187-197.     July,  1904. 
Nos.  20  and  21  in  one  cover .25 

22.  On  the  Toxicity  of  Distilled  Water  for  the  Fresh-water  Gammarus. 

Suppression  of  this  Toxicity  by  the  Addition  of  small  quantities  of 

Sodium  Chloride,  by  G.  Bullot.    Pp.  199-217.    July,  1904 .20 

Vol.2.  1.  The  Control  of  Heliotropic  Reactions  in  Fresh-water  Crustaceans  by 
Chemicals,  especially  CO3  (a  preliminary  communication),  by 
Jacques  Loeb.  Pp.  1-3.  November,  1904 „ 05 

2.  Further  Experiments  on  Heterogeneous  Hybridization  in  Echinodenns, 
by  Jacques  Loeb.  Pp.  5-30.  December,  1904 

8.  Influence  of  Calcium  and  Barium  on  the  Secretory  Activity  of  the 
Kidneys  (second  communication),  by  John  Bruce  MacCallum. 
Pp.  31-42.  December,  1904. 

4.  Note    on    the    Galvanotropic    Reactions    of    the   Medusa    Poly  orchis 
penicillata  A.  Agassiz,  by  Frank  W.  Bancroft.    Pp.  43-46.    Decem- 
ber, 1904. 
Nos.  2,  3  and  4  in  one  cover .45 

6.  The  Action  on  the  Intestines  of  Solutions  containing  two  Salts,  by 
John  Bruce  MacCallum.  Pp.  47-64.  January,  1905. 

6.  The   Action   of   Purgatives  in   a  Crustacean   (Sida   crystallina),   by 

John  Bruce  MacCallum.    Pp.  65-70.    January,  1905. 
Nos.  5  and  6  in  one  cover .25 

7.  On  the  Validity  of  Pfluger's  Law  for  the  Galvanic  Action  of  Para- 

mecium  .  (preliminary    communication),    by    Frank    W.    Bancroft. 
P.  71.    February,  1905. 

8.  On   Fertilization,   Artificial   Parthenogenesis   and  *  Cytolysis    of   the 

Sea-urchin  Egg,  by  Jacques  Loeb.  Pp.  73-81.    February,  1905. 

Nos.  7  and  8  in  one  cover , „      J.5 

9.  On  an  Improved  Method  of  Artificial  Parthenogenesis,  by  Jacques 

Loeb.     Pp.  83-86.     February,  1905 .05 

10.  On  the  Diuretic  Action  of  Certain  Haemolytics,  and  the  Action  of 

Calcium  in  Suppressing  Haemoglobinuria  (preliminary  communica- 
tion), by  John  Bruce  MacCallum.    Pp.  87-88.    March,  1905. 

11.  On  an  Improved  Method  of  Artificial  Parthenogenesis  (second  com- 

munication), by  Jacques  Loeb.    Pp.  89-92.    March,  1905. 
Nos.  10  and  11  in  one  cover 05 

12.  The  Diuretic  Action  of  Certain  Haemolytics  and  the  Influence  of 

Calcium  and  Magnesium  in  Suppressing  the  Haemolysis    (second 
communication),  by  John  Bruce  MacCallum.  Pp.  93-103.  May,  1905. 

13.  The  Action  of  Pilocarpine  and  Atropin  on  the  Flow  of  Urine,  by 

John  Bruce  MacCallum.    Pp.  105-112.    May,  1905. 
Nos.  12  and  13  in  one  cover 25 

14.  On  an  Improved  Method  of  Artificial  Parthenogenesis   (third  com- 

munication), by  Jacques  Loeb.    Pp.  113-123.     May,  1905 15 

15.  On  the   Influence   of  Temperature   upon   Cardiac   Contractions   and 

its  Relation  to  Influence  of  Temperature  upon  Chemical  Reaction 
Velocity,  by  Charles  D.  Snyder.    Pp.  125-146.    September,  1905 .25 

16.  Artificial  Membrane  Formation  and  Chemical  Fertilization  in  a  Star- 

fish (Asterina),  by  Jacques  Loeb.    Pp.  147-158.    September,  1905 15 

17.  On  the  Influence  of  Electrolytes  upon  the  Toxicity  of  Alkaloids  (pre- 

liminary communication),  by  T.  Brailsford  Robertson.    Pp.  159-162. 
October,  1905  05 

18.  Studies    on    the    Toxicity    of    Sea-water    for    Fresh-water    Animals 

(Gammarus   pulex   De   Geer),   by   C.   H.   Wolfgang   Ostwald.     Pp. 
163-191;   plates  1-6.     November,  1905 .35 

19.  On  the  Validity  of  Pfliiger's  Law  for  the  Galvanotropic  Reactions 

of  Paramecium*  by  Frank  W.  Bancroft.    Pp.  193-215;  8  text  figures. 
November,   1905  „.      .20 

Vol.3.  1.  On  Chemical  Methods  by  which  the  Eggs  of  a  Mollusc  (Lottia 
Gigantea)  can  be  caused  to  become  Mature,  by  Jacques  Loeb. 
Pp.  1-8.  November,  1905 05 

2.  On  the  Changes  in  the  Nerve  and  Muscle  which  seem  to  Underlie  the 

Electrotonic  Effect  of  the  Galvanic  Current,  by  Jacques  Loeb.    Pp. 

9-15.     December,  1905 05 

3.  Can  the  Cerebral  Cortex  be  Stimulated  Chemically?  .    (Preliminary" 

communication),  by  S.  S.  Maxwell.    Pp.  17-19.    February,  1906.. .05 

4.  The   Control   of  Galvanotropism  in   Paramecium   by   Chemical   Sub- 

stances, by  Frank  W.  Bancroft.    Pp.  21-23.    March,  1906 „  .10 

5.  The  Toxicity  of  Atmospheric  Oxygen  for  the  Eggs  of  the  Sea-urchin 

(Strsngyloeentrotus   purpuratus)    after   the    Process    of   Membrane 
Formation,  by  Jacques  Loeb.    Pp.  33-37.    March,  1906. 


UNTVEESITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

6.  On  the  Necessity  of  the  Presence  of  Free  Oxygen  in  the  Hypertonic 

Sea-water   for   the   Production   of   Artificial   Parthenogenesis,   by 
Jacques  Loeb.    Pp.  39-47.    March,  1906. 
Nos.  5  and  6  in  one  cover 15 

7.  On  the  Counteraction  of  the  Toxic  Effect  of  Hypertonic  Solutions 

upon  the  Fertilized  and  Unfertilized  Egg  of  the  Sea-urchin  by  Lack 

of  Oxygen,  by  Jacques  Loeb.    Pp.  49-56.    April,  1906... .05 

8.  On  the  Production  of  a  Fertilization  Membrane  in  the  Egg  of  the 

Sea-urchin  with  the  Blood  of  Certain  Gephyrean  Worms   (a  pre- 
liminary note),  by  Jacques  Loeb.    Pp.  57-58.    March,  1907 „.      .05 

9.  Note  on  the  Synthesis  of  a  Protein  through  the  Action  of  Pepsin 

(preliminary   communication),   by   T.   Brailsford   Robertson.     Pp. 
59-60.     April,  1907 ;..... „       .05 

10.  The  Chemical  Character  of  the  Process  of  Fertilization,  and  its  Bear- 

ing upon  the  Theory  of  Life-Phenomena,  by  Jacques  Loeb.     Pp. 
61-80.     September,  1907 25 

11.  A  New  Proof  of  the  Permeability  of  Cells  for  Salts  or  Ions  (a  pre- 

liminary communication),  by  Jacques  Loeb.    Pp.  81-86.     January, 

1908 „       .05 

12.  The  Origin  of  two  new  Retrogressive  Varieties  by  one  Mutation  in 

Mice,  by  Arend  L.  Hagedocrn.    Pp.  87-90.    September,  1908 05 

IS.  On  Synthesis  of  Paranuclein  through  the  Agency  of  Pepsin  and  Chemi- 
cal Mechanics  of  Hydrolysis  and  Synthesis  of  Proteins  through  the 
Agency  of  Enzymes,  by  T.  B.  Eobertson.  Pp.  91-94.  December, 
1908  05 

14.  The  Inheritance  of  Yellow  Color  in  Eodents,  by  Arend  L.  Hagedoorn. 

Pp.  95-99.     March,  1909 05 

15.  Table  of  H+  and  OH~  Concentrations  corresponding  to  Electromotive 

Forces  determined  in  Gas-chain  measurements,  by  C.  L.  A.  Schmidt. 

Pp.  101-113.     September,  1909 10 

16.  The  Proteins,  by  T.  Brailsford  Eobertson.    Pp.  115-194.    October,  1910  $1.00 

17.  Further  Proof  of  the  Identity  of  Heliotropism  in  Animals  and  Plants, 

by  Jacques  Loeb  and  S.  S.  Maxwell.     Pp.  195-197.     January,  1910      .05 
Vol.4.      1.  Experiments  on  the  Function  of  the  Internal  Ear,  by  S.  S.  Maxwell. 

Pp.  1-4.    September,  1910  05 

2.  On  the  Eise  of  Temperature  in  Eabbits,  Caused  by  the  Injection  of  Salt 

Solutions,  by  Theo.  C.  Burnett.    Pp.  5-7.    September,  1910  05 

3.  A  Biochemical  Conception  of  Dominance,  by  A.  E.  Moore.    Pp.  9-15. 

September,  1910   05 

4.  Galvanotropic  Orientation  in  Gonium  pectorale,  by  A.  E.  Moore  and  T. 

H.  Goodspeed.    Pp.  17-23.    May,  1911  05 

5.  On  a  Possible  Source  of  the  Biological  Individuality  of  the  Tissues  and 

Tissue-fluids  of  Animal  Species,  by  T.  Brailsford  Eobertson.     Pp. 
25-30.     May,  1911  05 

6.  Some  Factors  Influencing  the   Quantitative  Determination  of  Gliadin, 

by  J.  E.  Greaves.    Pp.  31-74.    August,  1911  40 

7.  Errors  of  Eefraction  Occurring  in  the  Students  of  the  University  of 

California,  by  Theo.  C.  Burnett.    Pp.  75-77.    August,  1911  05 

8.  On  the  Cytolytic  Action   of  Ox-Blood  Serum  upon   Sea-Urchin  Eggs, 

and  Its  Inhibition  by  Proteins   (Preliminary  communication),  by  T. 

Brailsford  Eobertson.     Pp.  79-88.     February,  1912 10 

9.  On  the  Nature  of  the  Cortical  Layer  in  Sea  Urchin  Eggs,  by  A.  E. 

Moore.    Pp.  89-90.    March,  1912. 

10.  On  the  Nature  of  the  Sensitization  of  Sea  Urchin  Eggs  by  Strontium 
Chloride,  by  A.  E.  Moore.  Pp.  91-93.  March,  1912. 

Nos.  9  and  10  in  one  cover .05 

11.  On  the  Isolation  of  Ob'cytase,  the  Fertilizing  and  Cytolyzing  Substance 
in  Mammalian  Blood  Sera,  by  T.  Brailsford  Eobertson.  Pp.  95-102. 
March,  1912. 

12.  On  the  Extraction  of  a  Substance  from  the  Sperm  of  a  Sea  Urchin 

(Strongylocentrotus  purpuratus)  which  will  Fertilize  the  Eggs  of  that 
Species,  by  T.  Brailsford  Eobertson.    Pp.  103-105.    March,  1912. 

13.  The  Demonstration  of  "Masked"  Iron  in  Blood,  by  C.  B.  Bennett. 

Pp.  107-108.    March,  1912. 

Nos.  11,  12  and  13  in  one  cover  10 

14.  A  New  Method  of  Heterogeneous  Hybridization  in  Echinoderms,  by 

A.  E.  Moore.    Pp.  109-110.    March,  1912. 

15.  Can  the  Presence  of  Acid  Account  for  the  Oedema  of  Living  Muscle, 

by  A.  E.  Moore.     Pp.  111-114.    April,  1912. 
Nos.  14  and  15  in  one  cover  ..: 05 

16.  On  the  Oxydations  and  Cleavages  of  Glucose.     Yeast  Glucase,  a  New 

Glucolytic  Ferment,  by  Victor  Birckner.     Pp.  115-183.     September, 

1912 75 

Other  series:  American  Archaeology  and  Ethnology,  Botany,  Classical  Philology,  Eco- 
nomics, Engineering,  Entomology,  Geology,  History,  Lick  Observatory  Bulletins,  Lick  Ob- 
servatory Publications,  Mathematics,  Modern  Philology,  Pathology,  Philosophy,  Psychology* 
Publications  of  the  Academy  of  Pacific  Coast  History,  and  Zoology. 


NON-CIRCUUTING  BOOK 


